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1

Electronic Resource

Accelerated production of transgenic wheat (Triticum aestivum L.) plants (1996)

Altpeter, Fredy ; Vasil, Vimla ; Srivastava, Vibha ; [et al.]

Springer

Plant cell reports 16 (1996), S. 12-17

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ISSN: 1432-203X

Keywords: Key words:Triticum aestivum L., wheat, transformation, biolistics.

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract. We have developed a method for the accelerated production of fertile transgenic wheat (Triticum aestivum L.) that yields rooted plants ready for transfer to soil in 8–9 weeks (56–66 days) after the initiation of cultures. This was made possible by improvements in the procedures used for culture, bombardment, and selection. Cultured immature embryos were given a 4–6 h pre- and 16 h post-bombardment osmotic treatment. The most consistent and satisfactory results were obtained with 30 μg of gold particles/bombardment. No clear correlation was found between the frequencies of transient expression and stable transformation. The highest rates of generation and transformation were obtained when callus formation after bombardment was limited to two weeks in the dark, with or without selection, followed by selection during regeneration under light. Selection with bialaphos, and not phosphinothricin, yielded more vigorously growing transformed plantlets. The elongation of dark green plantlets in the presence of 4–5 mg/l bialaphos was found to be reliable for identifying transformed plants. Eighty independent transgenic wheat lines were produced in this study. Under optimum conditions, 32 transformed wheat plants were obtained from 2100 immature embryos in 56–66 days, making it possible to obtain R3 homozygous plants in less than a year.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/s002990050166

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2

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Plant transformation by particle bombardment of embryogenic pollen (1995)

Stöger, Eva ; Fink, Christine ; Pfosser, Martin ; [et al.]

Springer

Plant cell reports 14 (1995), S. 273-278

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ISSN: 1432-203X

Keywords: pollen embryogenesis ; particle gun ; ImaGeneGreen ; Nicotiana tabacum

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Summary Direct delivery of DNA into embryogenic pollen was used to produce transgenic plants in tobacco. A plasmid bearing the ß-glucuronidase (GUS) marker gene in fusion with the 35S-promoter was introduced by microprojectile bombardment into mid-binucleate pollen of Nicotiana tabacum that had been induced to form embryos by a starvation treatment. In cytochemical expression assays, 5 out of 104 pollen grains were GUS+. Visual selection by staining with a non-lethal substrate for GUS was used to manually isolate transformed embryos. From the initial population of embryogenic GUS+ pollen, 1–5% developed into multicellular structures and 0.02% formed regenerable embryos. Two haploid transformants were regenerated. GUS expression was detected in different parts of the plants, and Southern analysis confirmed stable integration of the foreign DNA. Diploidisation was induced by injection of colchicine into the stem near adventitious buds. Offspring from selfings and backcrosses of one transformant were tested for GUS expression and by Southern blots. All F1-plants were transgenic, in accordance with Mendelian inheritance.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00232027

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3

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Accelerated production of transgenic wheat (Triticum aestivum L.) plants (1996)

Altpeter, Fredy ; Vasil, Vimla ; Srivastava, Vibha ; [et al.]

Springer

Plant cell reports 16 (1996), S. 12-17

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ISSN: 1432-203X

Keywords: Triticum aestivum L. ; wheat ; transformation ; biolistics

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract We have developed a method for the accelerated production of fertile transgenic wheat (Triticum aestivum L.) that yields rooted plants ready for transfer to soil in 8–9 weeks (56–66 days) after the initiation of cultures. This was made possible by improvements in the procedures used for culture, bombardment, and selection. Cultured immature embryos were given a 4–6 h pre-and 16 h post-bombardment osmotic treatment. The most consistent and satisfactory results were obtained with 30 μg of gold particles/bombardment. No clear correlation was found between the frequencies of transient expression and stable transformation. The highest rates of regeneration and transformation were obtained when callus formation after bombardment was limited to two weeks in the dark, with or without selection, followed by selection during regeneration under light. Selection with bialaphos, and not phosphinothricin, yielded more vigorously growing transformed plantlets. The elongation of dark green plantlets in the presence of 4–5 mg/l bialaphos was found to be reliable for identifying transformed plants. Eighty independent transgenic wheat lines were produced in this study. Under optimum conditions, 32 transformed wheat plants were obtained from 2100 immature embryos in 56–66 days, making it possible to obtain R3 homozygous plants in less than a year.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF01275440

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4

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Transgenic pea seeds as bioreactors for the production of a single-chain Fv fragment (scFV) antibody used in cancer diagnosis and therapy (2000)

Perrin, Yolande ; Vaquero, Carmen ; Gerrard, Ian ; [et al.]

Springer

Molecular breeding 6 (2000), S. 345-352

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ISSN: 1572-9788

Keywords: carcinoembryonic antigen ; recombinant antibody ; single-chain Fv fragment ; seed storage ; transgenic pea

Source: Springer Online Journal Archives 1860-2000

Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition

Notes: Abstract Field pea (Pisum sativum L.) appears well suited for the production of high-value molecules such as recombinant antibodies, with well-established agricultural practices world-wide and seeds that are easily stored and distributed. In order to evaluate the suitability of this grain legume for the production of biologically active antibodies, we transformed peas with a cDNA encoding the single-chain Fv fragment scFvT84.66. This scFv is derived from the monoclonal antibody T84.66, which recognises the well-characterised tumour-associated carcinoembryonic antigen. The antibody is useful for in vitro immunodiagnosis and in vivo imaging of human cancers. We expressed scFvT84.66 cDNA under the control of the seed-specific legumin A promoter. We targeted the antibody to the endoplasmic reticulum for better stability and high accumulation. Transgenic plants produced up to 9 μg per gram fresh weight of functional scFvT84.66 in their seeds. The transgene was stably inherited and expressed in the progeny, and the antibody remained active after storage in dried transgenic seeds for two months at room temperature. Our results demonstrate the suitability of grain legume seeds to produce biologically active recombinant antibodies, and the utility of field pea seeds as production vehicles for recombinant pharmaceutical macromolecules.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1009657701588

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5

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Zur Permeabilität der Schließzellen (1950)

Stöger, Eva Maria

Springer

Protoplasma 39 (1950), S. 588-596

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ISSN: 1615-6102

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Zusammenfassung Die vorliegenden Versuche, die mit Hilfe einer von Reuter (1943) ausgearbeiteten Methode die Permeabilitätseigenschaften des Schließzellenplasmas vonTradescantia viridis prüften, brachten die folgenden Ergebnisse: 1. Die Höhe der Durchlässigkeit des Schließzellenplasmas ist in erster Linie abhängig von der Belichtung, wird aber auch vom Feuchtigkeitsgrad beeinflußt. Maximale Permeabilität finden wir bei der Verbindung von photoaktiver und hydroaktiver Öffnungstendenz. 2. In denjenigen Fällen, in denen die beiden Reaktionssysteme der Spaltöffnungsbewegungen nicht gleichsinnig, sondern gegensinnig verlaufen (photoaktives Öffnen und hydroaktives Schließen und umgekehrt), zeigen die Schließzellen verminderte Durchlässigkeit. 3. Was die Art der permeierenden Stoffe anlangt, zeigen die Schließzellen a) für Acetamid unter allen Versuchsbedingungen die höchste Permeabilität, b) für Glyzerin die geringste Durchlässigkeit, c) für alle übrigen verwendeten Stoffe (Harnstoff, Urotropiu, Sulfoharnstoff, Methylharnstoff) ein mittleres Permeationsvermögen.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF01247686

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6

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Cereal crops as viable production and storage systems for pharmaceutical scFv antibodies (2000)

Stöger, Eva ; Vaquero, Carmen ; Torres, Esperanza ; [et al.]

Springer

Plant molecular biology 42 (2000), S. 583-590

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ISSN: 1573-5028

Keywords: transgenic cereals ; molecular pharming ; antibodies ; carcinoembryonic antigen ; single-chain Fv fragment ; particle bombardment

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract This report describes the stable expression of a medically important antibody in the staple cereal crops rice and wheat. We successfully expressed a single-chain Fv antibody (ScFvT84.66) against carcinoembryonic antigen (CEA), a well characterized tumor-associated marker antigen. scFv constructs were engineered for recombinant antibody targeting to the plant cell apoplast and ER. Up to 30 μg/g of functional recombinant antibody was detected in the leaves and seeds of wheat and rice. We confirmed that transgenic dry seeds could be stored for at least five months at room temperature, without significant loss of the amount or activity of scFvT84.66. Our results represent the first transition from model plant expression systems, such as tobacco and Arabidopsis, to widely cultivated cereal crops, such as rice and wheat, for expression of an antibody molecule that has already shown efficacy in clinical applications. Thus, we have established that molecular pharming in cereals can be a viable production system for such high-value pharmaceutical macromolecules. Our findings provide a strong foundation for exploiting alternative uses of cereal crops both in industrialized and developing countries.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1006301519427

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7

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Rice cell culture as an alternative production system for functional diagnostic and therapeutic antibodies (1999)

Torres, Esperanza ; Vaquero, Carmen ; Nicholson, Liz ; [et al.]

Springer

Transgenic research 8 (1999), S. 441-449

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ISSN: 1573-9368

Keywords: CEA ; KDEL ; molecular pharming ; recombinant antibody ; single chain Fv fragment ; transgenic rice

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract We investigated the suitability of transformed rice cell lines as a system for the production of therapeutic recombinant antibodies. Expression constructs encoding a single-chain Fv fragment (scFvT84.66, specific for CEA, the carcinoembryonic antigen present on many human tumours) were introduced into rice tissue by particle bombardment. We compared antibody production levels when antibodies were either secreted to the apoplast or retained in the endoplasmic reticulum (ER) using a KDEL retention signal. Production levels were up to 14 times higher when antibodies were retained in the ER. Additionally, we compared constructs encoding different leader peptides (plant codon optimised murine immunoglobulin heavy and light chain leader peptides) and carrying alternative 5′ untranslated regions (the petunia chalcone synthase gene 5′ UTR and the tobacco mosaic virus omega sequence). We observed no significant differences in antibody production levels among cell lines transformed with these constructs. The highest level of antibody production we measured was 3.8 μg g−1 callus (fresh weight). Immunological analysis of transgenic rice callus confirmed the presence of functional scFvT84.66. We discuss the potential merits of cell culture for the production of recombinant antibodies and other valuable macromolecules.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1008969031219

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8

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Constitutive Expression of Soybean Ferritin cDNA Intransgenic Wheat and Rice Results in Increased Iron Levels in Vegetative Tissues but not in Seeds (2000)

Drakakaki, Georgia ; Christou, Paul ; Stöger, Eva

Springer

Transgenic research 9 (2000), S. 445-452

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ISSN: 1573-9368

Keywords: ferritin ; transgenic rice ; transgenic wheat ; iron

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract We used particle bombardment to produce transgenic wheat and rice plants expressing recombinant soybean ferritin, a protein that can store large amounts of iron. The cDNA sequence was isolated from soybean by RT-PCR and expressed using the constitutive maize ubiquitin-1 promoter. The presence of ferritin mRNA and protein was confirmed in the vegetative tissues and seeds of transgenic wheat and rice plants by northern and western blot analysis, respectively. The levels of ferritin mRNA were similar in the vegetative tissues of both species, but ferritin protein levels were higher in rice. Both ferritin mRNA and protein levels were lower in wheat and rice seeds. ICAP spectrometry showed that iron levels increased only in vegetative tissues of transgenic plants, and not in the seeds. These data indicate that recombinant ferritin expression under the control of the maize ubiquitin promoter significantly increases iron levels invegetative tissues, but that the levels of recombinant ferritin in seeds are not sufficient to increase iron levels significantly over those in the seeds of non-transgenic plants.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1023/A:1026534009483

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