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     

Comparison of different strains ofCellulomonas for production of cellulolytic and xylanolytic enzymes from biomass produced on saline lands

A comparison of the rate of carboxymethyl cellulase (CMCase), avicelase, xylanase, -glucosidase and -xylosidase production and rates of growth by 4 different strains ofCellulomonas revealed a wide range of behaviour; with some strains producing more CMCase, avicelase, xylanase, -glucosidase and -xylosidase from complex lignocellulosic (LC) biomass (from saline land) and CMC while some others producing small amounts of these enzymes. One strain,C. biazotea, was better with respect to enzyme production potential and growth behaviour than most of the other strains and has been chosen as a starting strain for genetic improvement for producing enzymes of the cellulase complex.     

Synthesis and assembly of soybean β-conglycinin in vitro

The construction of SP6-derived expression plasmids that encode normal and modified -conglycinin subunits is described. With the exception of an additional methionine at their NH₂-terminal ends and the lack of glycans, the normal subunits synthesized at the direction of these plasmids coresponded to mature and subunits isolated from soybean seeds. The subunits assembled into trimers in vitro that were equivalent in size to those formed in vivo. This result shows that the glycans are not required either for protein folding or oligomer assembly. Subunits produced from other plasmids, which had modifications in a highly conserved hydrophobic region in the COOH-terminal end of the subunits, either did not assemble or assembled at an extremely low rate compared to unmodified subunits. Structural changes at the more hydrophilic NH₂-terminal end had mixed effects. Several subunits modified in this region assembled into trimers at rates that were either equal or greater than those for normal subunits. Others assembled less completely than the normal subunits. Our results indicate that the in vitro synthesis and assembly assay will be useful in evaluating structure-function relationships in modified -conglycinin subunits. The results also show that structural changes at the NH₂-terminal end of the subunits are tolerated to a greater extent than modifications in the hydrophobic conserved region in the COOH-terminal half of the subunits, and this information will be useful in efforts to improve soybean quality.     

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     

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     

Organogenesis and plant regeneration from immature embryos of Rosa hybrida L.

Intact, flowering, rose plants have been regenerated in vitro from excised embryos of crosses between Bridal Pink (the maternal parent) and several pollen parents. Explanted embryonic tissues developed into an organogenic callus which formed adventitious shoots after several months only on a modified half-strength Murashige & Skoog medium containing 1.0 M BA and 0.05 M NAA. These shoots could be separated, grown individually, rooted in a medium with no BA or NAA, with 1.0 M IBA, and transplanted to greenhouse media. Embryos ranging in age from 21 to 35 days post-pollination formed organogenic callus that eventually regenerated adventitious shoots. Histological examination of normally-developing embryos showed that well-defined embryonic axes were beginning to develop at approximately 20–25 days postpollination. Analysis of populations of regenerated plants from different crosses showed differences in flower color, growth habit, peduncle length, and petal number. This system may be useful for irradiation-mutation breeding and/or for the development of transgenic rose plants using Agrobacterium tumefaciens.Abbreviations BA 6-benzyladenine - IAA 3-indoleacetic acid - IBA 3-indolebutanoic acid - MS Murashige & Skoog - NAA -naphthaleneacetic acid - 2iP N⁶-(2-isopentenyl)adenosine     

Shoot and root organogenesis of Camellia sasanqua

In vitro-derived shoot tips, (10 mm) taken from primary cultures of Camellia sasanqua L., were evaluated for organogenesis when cultured on a half-strength MS medium supplemented with various concentrations of NAA, IBA, BA and GA₃. Maximum shoot proliferation and growth for juvenile and mature tissue was obtained when 0.54 M NAA, 8.8 M BA plus 14.4 to 28.9 M GA₃ was added to the culture media, with a pH between 4.5 and 5.0. In vitro-derived shoots (20 mm) from mature C. sasanqua Day Dream and juvenile C. sasanqua cultures initiated roots in vitro after immersion in 2.5 mM IBA for 30 min. Sixty percent of the mature shoots and 90% of the juvenile shoots initiated roots within 3 weeks of treatment with IBA.Abbreviations MS Murashige and Skoog (1962) - IBA lH-indole-3-butanoic acid - BA N-(phenyl-methyl)-lH-purine-6-amine - GA₃ gibberellic acid - kinetin N-(Z-furanyl-methyl)-lH-purine-6-amine - NAA l-naphthaleneacetic acid - L Linear - Q Quadratic     

Comparative study of the antitumor effect of two types of murine recombinant interferons, (β) and (γ), against B16-F10 melanoma

Summary A comparative study of the antitumor effect of murine recombinant interferon() Mu-rIFN() and murine recombinant interferon() Mu-rIFN() on B16-F10 melanoma was conducted. Administration of Mu-rIFN() i.p. into C57BL/6 mice on days 1 to 7 produced a higher suppressive effect than Mu-rIFN() both on the growth of s.c. implanted tumor and on the formation of artificial pulmonary metastasis. Pharmacokinetic study of Mu-rIFN() demonstrated that high plasma levels were retained for a long time. In clonogenic assay, Mu-rIFN() at 1000 units/ml showed about 80% inhibition of colonies of B16-F10 melanoma. However, Mu-rIFN() hardly inhibited the colonies, even at 1000 units/ml. Augmentation of natural killer (NK) cytotoxicity was much greater with Mu-rIFN() than Mu-rIFN(), whereas Mu-rIFN() enhanced the cytotoxicity of peritoneal macrophages more strongly than Mu-rIFN(). Injection of Mu-rIFN() i.p. 1 day before tumor challenge also inhibited the formation of pulmonary metastasis of B16-F10 melanoma. However, pretreatment of mice with carrageenan significantly suppressed the inhibitory effect of Mu-rIFN(). From these results, it is suggested that the inhibitory effect of Mu-rIFN() on the tumor growth and metastases of B16-F10 melanoma is mediated partly by direct antitumor effect and partly by the activation of macrophages, and that the augmentation of NK activity contributes mainly to the antitumor effect of Mu-rIFN().     

The promoter of aBrassica napus polygalacturonase gene directs pollen expression ofβ-glucuronidase in transgenicBrassica plants

A 647-bp 5-flanking fragment obtained from genomic clone Sta 44G(2) belonging to a family of polygalacturonase genes expressed inBrassica napus pollen was fused to the-glucuronidase (GUS) marker gene. This fusion construct was introduced intoB. napus plants viaAgrobacterium tumefaciens transformation. Analysis of the transgenicB. napus plants revealed that this promoter fragment is sufficient to direct GUS expression specifically in the anther and that GUS activity increases in pollen during maturation.Abbreviation GUS -Glucuronidase     

Factors influencing Agrobacterium tumefaciens-mediated transformation and regeneration of the safflower cultivar ‘centennial’

The effects of co-cultivation conditions on transformation efficiency and direct shoot regeneration from seedling explants of safflower cv. Centennial were examined. Agrobacterium tumefaciens strain EHA105/p35SGUSInt was more infective than LBA4404/pBI121 as determined by numbers of sectors expressing -glucuronidase activity. Compared to nontransformed controls, efficiency of direct shoot regeneration was markedly decreased by co-cultivation with EHA105 and the decrease exacerbated by addition of acetosyringone, indicating that a hypersensitive response to bacterial infection may reduce organogenetic potential. Likewise exposure of co-cultivated explants to kanamycin or geneticin in selective media reduced regeneration efficiency. Addition of 500 mg l^-1 carbenicillin slightly increased numbers of regenerating shoots. Tranfformed shoots were obtained only when kanamycin selection was initiated 1 or 2 days after co-cultivation. Presence of transgenes in geneticin-resistant shoots was confirmed using polymerase chain reaction and Southern hybridization assays.Abbreviations AS acetosyringone - GUS -glucuronidase - MS Murashige and Skoog (1962) - NAA naphthaleneacetic acid - NPTII neomycin phosphotransferase II - PCR polymerase chain reaction - TDZ thidiazuron     

Genetic characterization of a new splice variant of the β2 subunit of the voltage-dependent calcium channel

This study reports a novel splice variant form of the voltage-dependent calcium channel ₂ subunit (_2g). This variant is composed of the conserved amino-terminal sequences of the _2a subunit, but lacks the -subunit interaction domain (BID), which is thought essential for interactions with the ₁ subunit. Gene structure analysis revealed that this gene was composed of 13 translated exons spread over 107 kb of the genome. The gene structure of the ₂ subunit was similar in exon-intron organization to the murine ₃ and human ₄ subunits. Electrophysiological evaluation revealed that _2a and _2g affected channel properties in different ways. The _2a subunit increased the peak amplitude, but failed to increase channel inactivation, while _2g had no significant effects on either the peak current amplitude or channel inactivation. Other subunits, such as ₃ and ₄, significantly increased the peak current and accelerated current inactivation.     

In vitro regeneration through organogenesis of Meconopsis simplicifolia — An endangered ornamental species

Meconopsis simplicifolia (D.Don) Walp. could be propagated by induction of adventitious shoots from callus produced on hypocotyl, cotyledon and rosette leaf explants of 4-month-old seedlings. Callus was initiated on agar-solidified Murashige and Skoog medium supplemented with 1 M kinetin +10 M -naphthaleneacetic acid (NAA). Shoots formed when the callus was subcultured on medium supplemented with kinetin or benzyladenine (BA) in combination with NAA, indoleacetic acid, indolebutyric acid or gibberellic acid. Excised shoots were rooted on medium containing auxin with 10 M NAA producing the best rooting (55%).Abbreviations BA 6-benzyladenine - 2,4-d 2,4-dichlorophenoxy-acetic acid - FAA formalin-acetic acid-alcohol - GA₃ gibberellic acid - IAA indole-3-acetic acid - IBA -indolebutyric acid - NAA -naphthaleneacetic acid     

Identification of a specific protein in the mitochondrial fraction of the rat heart whose phosphorylation is inhibited by taurine

Summary It was previously reported that the mitochondrial fraction of the rat heart contained a specific protein with a molecular weight of approximately 44kDa whose phosphorylation was inhibited by taurine (Lombardini,1994a). Isolation of the 44kDa phosphoprotein on a 1-dimensional polyacrylamide gel using traditional glycine buffers followed by re-electrophoresing the cut out proportion of the gel which corresponds to the 44kDa protein on a tricine-buffered gel resulted in sufficient pure protein for sequence analysis. The results indicate that the 44kDa phosphoprotein is pyruvate dehydrogenase.     

In vitro meristem cloning of Eucalyptus tereticornis Sm.

Rapid multiplication of axillary meristems and direct shoot development occurred from nodal explants of mature Eucalyptus tereticornis Sm. with 5.3 M NAA, 1.1 M IAA and 4.4 M BA in Murashige-Skoog medium. Repeated subcultures of the second generation shoot cultures into low cytokinin-auxin containing media (0.44–0.88 M BA+0.1 M NAA) yielded axillary microshoots in large numbers. Half-strength MS liquid medium with 4.9 M IBA, 5.5 M IAA and 5.3 M NAA for four days, half-strength semi-solid hormonefree MS medium with charcoal, and MS liquid medium without charcoal and hormones, in sequence, induced rooting of shoots in the dark. This system is suitable for the mass propagation of this difficult-to-root eucalypt.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic acid - IBA -indole-3-butyric acid - 2-iP isopentyl adenine - Kn kinetin - MS Murashige-Skoog - NAA -naphthalene acetic acid - PVP polyvinylpyrrolidone     

Agrobacterium tumefaciens-mediated transformation of safflower (Carthamus tinctorius L.) cv. ‘Centennial’

Efficient callus formation was achieved from cotyledon, stem, and leaf expiants of the domestic safflower cultivar Centennial on MS salts medium containing 1 mg/L BAP and 1 mg/L NAA. Shoot buds were regenerated from 26% of leaf-derived calli on callus induction medium, although attempts to root regenerated shoots were not successful. Centennial expiants inoculated with Agrobacterium tumefaciens containing NPT II and GUS genes produced kanamycin-resistant calli from which buds were regenerated. Transformation and stable integration of transgenes was confirmed by GUS assay and DNA hybridization in kanamycin-resistant calli, and GUS assay in regenerated shoots.Abbreviations BAP 6-benzylaminopurine - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - GUS -glucuronidase - IAA indole-3-acetic acid - NPT II neomycin phosphotransferase II     

Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation

An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90–100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO₃ and 0.5–2 mg/L BA in combination with 0.01–0.05 mg/L 2,4-D or 0.1–1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All R_o transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R₁ seed progeny and showed co-segregation.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - NPTII neomycin phosphotransferase type II - GUS -glucuronidase - CaMV cauliflower mosaic virus - MS Murashige and Skoog - X-Gluc 5-bromo-4-chloro-3-indolyl-D--glucuronic acid - IBA indolebutyric acid - SDS sodium dodecyl sulfate     

Testis-specific β2 tubulins are identical in Drosophila melanogaster and D. hydei but differ from the ubiquitous β1 tubulin

In Drosophila as in many organisms tubulins are encoded by a gene family. We have determined the complete nucleotide sequences coding for the 1 and 2 tubulins of Drosophila melanogaster and the 2 tubulin of D. hydei, and found these insect tubulins to be highly conserved and like tubulins of other organisms. This is discussed with reference to the possible functional domains of these proteins. — The 1 tubulin gene of Drosophila is constitutively expressed, whereas the 2 tubulin is expressed specifically in the testes. In D. melanogaster the amino acid sequences of these proteins are 95% homologous, differing at only 25 positions. In the testes the 2 tubulin participates in different microtubules as shown by genetic analysis (Kemphues et al. 1982). Interestingly, all of the amino acids characteristic of the testis-specific 2 tubulin are also present in the corresponding gene of D. hydei. Of special interest is the high degree of conservation of the carboxy-terminal domain in these functionally equivalent tubulins.     

Regeneration of plantlets from cell suspension culture derived callus of white poplar (Populus alba L.)

Friable calli derived from the stem tissues of Populus alba were used to establish cell suspension cultures which were characterized for in vitro growth and regeneration capacity. Suspended cells and callus recovered from these cells were maximal on a fresh weight basis using MS liquid medium containing 0.44 M BAP and 4.52 M 2,4-D. Shoot regeneration from the recovered callus was observed within 30 to 40 days of culture. The number of shoots was increased by subculturing the shoot-forming callus 2 to 3 times on MS medium supplemented with 19.7 M 2iP and 0.05 M IBA. Regenerated shoots were easily rooted on half-strength MS medium lacking growth regulators, and the plantlets were transferred to pots containing vermiculite for greenhouse growth.Abbreviations BAP 6-benzylaminopurine - 2iP 2-isopentenyladenine - NAA 1-naphthaleneacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - 2,4-D 2,4-dichlorophenoxyacetic acid - PCV packed cell volume - MS medium Murashige and Skoog medium (1962)     

Einige Bemerkungen über die Ersatzgeschlechtstiere vonReticulitermes

Zusammenfassung An Laboratoriumszuchten vonReticulitermes lucifugus Rossi undReticulitermes flavipes Kollar (aus den Hamburger Befallsgebieten) werden Beobachtungen zur Biologie der Ersatzgeschlechtstiere mitgeteilt. Für die Durchführung dieser Beobachtungen wurden besondere Schaunester eingerichtet. Folgende Punkte der beobachteten Verhaltensweisen werden hier hervorgehoben: Die Nymphen der Ersatzgeschlechtstiere sammeln sich stets in Herden von 80–100 Einzeltieren und werden von einer Gruppe Arbeiter (als Hütehunde) bewacht. Die Arbeiter halten die Nymphen auf einem engen Raum zusammen und versuchen andere Nestgenossen von den Nymphen fernzuhalten. Alle ein bis zwei Tage wechseln die Herden ihren Standort. Das Weiterziehen geschieht gleichfalls in Form von Herden.Die Ernährung der Nymphen geschieht auf stomodealem Wege durch die Arbeiter. *** DIRECT SUPPORT *** A0180089 00007     

Effects of G-protein probes on interactions between auxin efflux carriers and phytotropin receptors in zucchini (Cucurbita pepo L.) microsomal membranes

Microsomal vesicles prepared from etiolated hypocotyl tissue of zucchini (Cucurbita pepo L. cv. All Green Bush) exhibited saturable N-1-naphthylphthalamic acid ([³H]NPA) binding, NPA-stimulated association of indol-3yl-acetic acid ([³H]IAA), and saturable binding of guanosine 5-O-[3-thiotriphosphate] (GTP--[³⁵S]). These vesicles were used to test the possibility that NPA receptors might interact with IAA-anion efflux carriers by coupling through a GTP-binding protein (G-protein). Unlabelled GTP--S or guanosine 5-O-[2-thiodiphosphate] (GDP--S) had no effect on saturable NPA binding or on the NPA-stimulated association of IAA with microsomes. NPA did not affect saturable binding of GTP--[³⁵S] to microsomes, either in the presence or absence of saturating concentrations of unlabelled GTP--S or GDP. It is concluded that the occupancy of phytotropin receptors is not transduced to auxin efflux carriers by a GTP-binding protein.