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Stable co-transformation of maize protoplasts with gusA and neo genes (1989)

Lyznik, Leszek A. ; Ryan, Randy D. ; Ritchie, Steven W. ; [et al.]

Springer

Plant molecular biology 13 (1989), S. 151-161

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ISSN: 1573-5028

Keywords: β-glucuronidase (gusA) gene ; maize ; protoplasts ; stable co-transformation ; transformation ; Zea mays L.

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract An efficient co-transformation protocol using polyethylene glycol was developed for Zea mays L. (cv. A188 × BMS) protoplasts isolated from suspension culture cells. Co-transformation was accomplished by using plasmid constructions containing β-glucuronidase (gusA) or neomycin phosphotransferase (neo) gene coding sequences; both were under control of the CaMV 35S promoter. Protoplast culture and transformation conditions were optimized to assure efficient recovery of transformed cells. The overall efficiency of transformation was 1 × 10−4 (calculated per viable protoplast plated). Among kanamycin-resistant lines, 50% showed a high level of GUS activity (above one unit). Southern blot hybridization confirmed the presence of numerous gusA and neo coding sequences in the maize genome. In two analyzed lines, integrated sequences appeared to be organized in tandem head-to-tail repeats. Results also indicated that the integrated sequences were partially methylated.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00016134

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Identification of a maize root transcript expressed in the primary response to nitrate: characterization of a cDNA with homology to ferredoxin-NADP+ oxidoreductase (1994)

Ritchie, Steven W. ; Redinbaugh, Margaret G. ; Shiraishi, Naomasa ; [et al.]

Springer

Plant molecular biology 26 (1994), S. 679-690

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ISSN: 1573-5028

Keywords: primary response ; ferredoxin NADP+ oxidoreductase ; nitrate ; cycloheximide ; Zea mays ; roots

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract To more fully understand the biochemical and molecular events which occur in plants exposed to nitrate, cDNAs whose accumulation was enhanced in nitrate- and cycloheximide-treated maize (Zea mays L. W64A × W182E) roots were isolated. The 340 bp Zmrprn 1 (for Zea mays root primary response to nitrate) cDNA also hybridized with a probe enriched for nitrate-induced sequences, and was characterized further. Sequence analysis of a near full-length cDNA (Zmrprn 1A) showed strong homology (〉90% amino acid identity) with a root ferredoxin-NADP+ oxidoreductase (FNR) of rice, and 45–50% amino acid identity with leaf FNR genes. When expressed in Escherichia coli, the Zmrprn 1A cDNA produced a protein with NADPH: ferricyanide reductase activity, consistent with the enzymatic properties of an FNR. The Zmrprn 1 cDNA hybridized with a 1.4 kb transcript which was expressed in the maize root primary response to nitrate. That is, mRNA levels in roots increased rapidly and transiently in response to external nitrate, and low levels of nitrate (10 μM) induced transcript accumulation. The accumulation of the Zmrprn 1 transcript was not prevented by cycloheximide, indicating that the cellular factor(s) required for expression were constitutively present in maize roots. The Zmrprn 1 mRNA accumulated specifically in response to nitrate, since neither K+ nor NH4 + treatment of roots caused transcript accumulation. Maize leaves had about 5% of the transcript level found in roots, indicating a strong preference for expression of Zmrprn 1 in roots. Analysis of maize genomic DNA indicated the presence of only a single gene or very small gene family for the Zmrprn 1. Together, the data indicate that Zmrprn 1A encodes a nitrate regulated maize root FNR.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00013753

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Factors influencing Agrobacterium-mediated transient expression of gusA in rice (1992)

Li, Xiu-Qing ; Liu, Chang-Nong ; Ritchie, Steven W. ; [et al.]

Springer

Plant molecular biology 20 (1992), S. 1037-1048

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ISSN: 1573-5028

Keywords: Agrobacterium ; rice ; transformation ; Ti plasmid ; GUS

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract Transient expression of GUS in rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens was characterized using binary vectors containing gusA genes that express minimal (pKIWI105 and pCNL1) or no (p35S-GUS-INT and pCNL56) GUS activity in bacteria. Four-day old seedlings obtained from seeds or immature embryos of rice were cut into shoot, root, and seed remnants and inoculated with various strains of A. tumefaciens. Transient GUS expression events were quantitated histochemically by determining the frequency of explants exhibiting blue spots indicative of GUS at four to six days after cocultivation with A. tumefaciens. A. tumefaciens strains that did not contain the gusA gene (At643) or a Ti-plasmid (At563 and At657) did not elicit any blue staining characteristic of GUS activity. Several parameters were important in obtaining efficient transient expression of GUS in rice mediated by A. tumefaciens. The growth regulator 2,4-D inhibited GUS expression if present during the seed germination period, but the presence of 6 mg/1 2,4-D during cocultivation of the explants with A. tumefaciens slightly enhanced GUS expression efficiency. All 21 rice cultivars tested expressed GUS after co-cultivation with A. tumefaciens. The GUS expression frequency was highest amongst the indica cultivars. The frequencies of GUS expression in japonica cultivars and in Oryza glaberrima cultivars (grown primarily in Africa) were generally one-half to one-third the level found for indica varieties. Leaf explants were more susceptible to A. tumefaciens-facilitated GUS expression than were roots or seed remnants. The vir genes of an agropine-type Ti-plasmid of A. tumefaciens were most effective in directing transient GUS expression in rice, whereas those of a nopaline-type and an octopine-type plasmid were less effective. We have also found that the frequency of transient expression of GUS was higher with pBIN19 as the precursor cloning vector than with pEND4K as the precursor cloning vector. Reasons for differences in effectiveness of these binary vectors are discussed. Using the conditions described here, A. tumefaciens-mediated frequencies of transient GUS expression in four-day old shoots of several rice cultivars were routinely in excess of 50%.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00028891

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Agrobacterium tumefaciens-mediated expression ofgusA in maize tissues (1993)

Ritchie, Steven W. ; Lui, Chang-Nong ; Sellmer, James C. ; [et al.]

Springer

Transgenic research 2 (1993), S. 252-265

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ISSN: 1573-9368

Keywords: Agrobacterium tumefaciens ; maize ; GUS ; gusA/intron ; particle bombardment

Source: Springer Online Journal Archives 1860-2000

Topics: Biology

Notes: Abstract To develop a system forAgrobacterium-mediated transformation of maize (Zea mays L.), we have investigated histochemically the transient expression of β-glucuronidase (GUS) activity in maize seedling tissue segments using binary vectors that allow minimal (pKIWI105 and pCNL1) or undetectable (p35S-GUS-INT and pCNL56) levels of GUS activity inA. tumefaciens. Tissue segments from three- to five-day-old sterile seedlings of maize genotype A188 were inoculated withA. tumefaciens. Four days after inoculation, transient expression of GUS activity was found in mesocotyl segments originating from the intercalary meristem region. This GUS activity was specific to the vascular cylinder and was not found in the internal cortical or epidermal layers, nor was it found in mature mesocotyl tissue (segments 5 mm below the coleoptilar node). Transient GUS activity was also detected in leaf and coleoptile tissues of shoot segments, but not in the shoot apexper se or in leaves younger than the first leaf. Maize tissues inoculated withA. tumefaciens strains that harbourgusA-containing binary vectors but no Ti-plasmid did not show GUS activity, supporting evidence from previous work thatvir gene activity was essential for the observed GUS activity.A. tumefaciens strains containing different types of Ti-plasmids were also tested. A strain harbouring an agropine-type Ti-plasmid was the most effective for expressing GUS activity in mesocotyl segments, whereas a strain harboring a nopaline-type Ti-plasmid was most effective for expression of GUS activity in the apical meristem-containing segment. These results indicate that different interactions occurred between the differentA. tumefaciens strains and the susceptible plant tissues. Maize genotype specificity for GUS activity in mesocotyl tissues was observed; variations in the cocultivation medium had a profound effect on the frequency of expression of GUS activity.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF01968838

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