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We have developed a new plant vector system for repeated transformation (called MAT for multi-autotransformation) in which a chimeric ipt gene, inserted into the transposable element Ac, is used as a selectable marker for transformation. Selectable marker genes conferring antibiotic or herbicide resistance, used to introduce economically valuable genes into crop plants, have three major problems: (i) the selective agents have negative effects on proliferation and differentiation of plant cells; (ii) there is uncertainty regarding the environmental impact of many selectable marker genes; (iii) it is difficult to perform recurrent transformations using the same selectable marker to pyramid desirable genes. The MAT vector system containing the ipt gene and the Ac element is designed to overcome these difficulties. When tobacco leaf segments were transformed and selected, subsequent excision of the modified Ac produced marker-free transgenic tobacco plants without sexual crosses or seed production. In addition, the chimeric ipt gene could be visually used as a selectable marker for transformation of hybrid aspen (Populus sieboldii x Populus grandidentata). The chimeric ipt gene, therefore, is an attractive alternative to the most widely used selectable marker genes. The MAT vector system provides a promising way to shorten breeding time for genetically engineered crops. This method could be particularly valuable for fruit and forest trees, for which long generation times are a more significant barrier to breeding and genetic analysis

Improvement of wheat (Triticum aestivum) by biotechnological approaches is currently limited by a lack of efficient and reliable transformation methodology. In this report, we detail a protocol for transformation of a highly embryogenic wheat cultivar, Bobwhite. Calli derived from immature embryos, 0.5 to 1 mm long, were bombarded with microprojectiles coated with DNA containing as marker genes the bar gene, encoding phosphinothricin-resistance, and the gene encoding beta-glucuronidase (GUS), each under control of a maize ubiquitin promoter. The bombardment was performed 5 d after embryo excision, just after initiation of callus proliferation. The ability of plantlets to root in the presence of 1 or 3 mg/L of bialaphos was the most reliable selection criteria used to identify transformed plants. Stable transformation was confirmed by marker gene expression assays and the presence of the bar sequences in high molecular weight chromosomal DNA of the resultant plants. Nine independent lines of fertile transgenic wheat plants have been obtained thus far, at a frequency of 1 to 2 per 1000 embryos bombarded. On average, 168 d elapsed between embryo excision for bombardment and anthesis of the T0 plants. The transmission of both the resistance phenotype and bar DNA to the T1 generation verified that germline transformation had occurred

Three sugarbeet selections differing in cross-resistance to three classes of acetolactate synthase (ALS)-inhibiting herbicides have been developed using somatic cell selection. Sugarbeet selections resistant to imidazolinone herbicides, Sir-13 and 93R30B, do not metabolize [14C]-imazethapyr any faster or differently than sensitive, wild-type sugarbeets or a sulfonylurea-resistant/imidazolinone-sensitive selection, Sur. ALS specific activity from the three herbicide-resistant selections ranged from 73 to 93% of the wild-type enzyme extracts in the absence of herbicide, indicating enzyme overexpression was not a factor in resistance. Acetolactate synthase from Sir-13 plants showed a 40-fold resistance to imazethapyr but no resistance to chlorsulfuron or flumetsulam. Polymerase chain reaction amplification and sequencing of two regions of the ALS gene spanning all known sites for ALS-based herbicide resistance in plants indicated a single nucleotide change in the Sir-13 gene (G337 to A337) resulting in a deduced substitution of threonine for alanine at position 113 in the sugarbeet amino acid sequence. Sur ALS was not significantly resistant to imazethapyr, but was 1,000- and 50-fold resistant to chlorsulfuron and flumetsulam, respectively. Sur gene sequencing indicated a single nucleotide change (C562 to T562) resulting in a serine for proline substitution at position 188 of the ALS primary structure. The 93R30B nucleotide sequence indicated two mutations resulting in two deduced amino acid substitutions: threonine for alanine at position 113 plus serine for proline at position 188. The 93R30B double mutant incorporated the changes observed in each of the single mutants above and correlated with higher resistance levels to imazethapyr (> 1,000-fold), chlorsulfuron (4,300-fold), and flumetsulam (200-fold) at the ALS level than observed in either of the single mutants. 93R30B represents the first double mutant derived by a two-step selection process that incorporates two class-specific ALS-inhibitor resistance mutations to form a single broad cross-resistance trait. The interaction of the two altered amino acids is synergistic with respect to enzyme resistance vs. the resistance afforded by each of the individual mutations.

Pokeweed antiviral protein (PAP), a 29-kDa protein isolated from Phytolacca americana inhibits translation by catalytically removing a specific adenine residue from the 28S rRNA of eukaryotic ribosomes. PAP has potent antiviral activity against many plant and animal viruses, including human immunodeficiency virus. We describe here development of a positive selection system to isolate PAP mutants with reduced toxicity. In vitro translation in the presence or absence of microsomal membranes shows that PAP is synthesized as a precursor and undergoes at least two different proteolytic processing steps to generate mature PAP. The PAP cDNA was placed under control of the galactose-inducible GAL1 promoter and transformed into Saccharomyces cerevisiae. Induction of PAP expression was lethal to yeast. The PAP expression plasmid was mutagenized and plasmids encoding mutant PAP genes were identified by their failure to kill S. cerevisiae. A number of mutant alleles were sequenced. In one mutant, a point mutation at Glu-177 inactivated enzymatic function in vitro, suggesting that this glutamic acid residue is located at or near the catalytic site. Mutants with either point mutations near the N terminus or a nonsense mutation at residue 237 produced protein that was enzymatically active in vitro, suggesting that the toxicity of PAP is not due solely to enzymatic activity. Toxicity of PAP appears to be a multistep process that involves possibly different domains of the protein.

Stable genetic transformation of Picea mariana (black spruce) was obtained via particle bombardment into two target tissues, mature cotyledonary somatic embryos and suspensions from embryonal masses, with the Biolistic PDS-1000/He device. Seven transgenic embryogenic cell lines were obtained from the mature cotyledonary somatic embryos after secondary somatic embryogenesis from two different cell lines (R4F14 and 119794-014). The suspension cultures from embryonal masses produced five transgenic cell lines from one cell line (R4F14). Expression of the introduced β-glucuronidase (GUS) and neomycin phosphotransferase II (NPT II) genes was detected by histochemistry and fluorometry, and by ELISA in 10 of the lines. Two lines showed only NPT II gene expression. Four of the five lines obtained after bombardment of suspensions of embryonal masses showed lower levels of expression of GUS and NPT II. The integration of the foreign genes was confirmed by polymerase chain reaction analyses and Southern hybridization for GUS and NPT II, and complex hybridization patterns were observed. The 12 transgenic lines obtained had a typical embryogenic morphology and were capable of maturation and germination. Over 40 transgenic trees were regenerated from one of the transgenic lines, and they have a normal phenotype.

Cell suspension cultures of 'Peter Pears', a cultivar of Gladiolus x grandiflorus (Hort.), susceptible to the fungus Fusarium oxysporum f. sp. gladioli (Mass.), have been challenged with fusaric acid, one of the toxins produced by this pathogen. Selected cell-lines showed increased tolerance to the toxin and grew even on concentrations of fusaric acid up to 0.5 mM. When inoculated with a conidial suspension, the mycelial growth on selected cell-lines was limited compared to the control. Fusaric acid greatly reduced plant regeneration, and only two plants were obtained from the callus subcultured for a prolonged time on medium with fusaric acid. These plants have a significantly altered DNA content compared to the control. A reduced callus phase on toxin-containing medium, resulted in an improved regeneration and the DNA content of the regenerated plantlets was similar to the control. Some plants regenerated from these fusaric acid-insensitive cell-lines showed an increased tolerance to the toxin when cultured in vitro in presence of fusaric acid. The selected plants will be further tested for Fusarium-resistance once maturity is reached.

A selection procedure is described that isolated Al-resistant individuals from a sensitive rice (Oryza sativa L.) genotype. Somaclonal variation was used as the only source of variability and selection pressures were applied at both callus and regenerated plant levels. When 10 week-old, embryo-derived calli were submitted to different Al stresses for a period of 20 weeks. After this in vitro stress period, selected calli were grown during 18 weeks, without selection pressure, on regeneration media. Selection pressures were applied on regenerated plants (R0), and 9 R0 plants which produced seeds were selected. The transmission of the Al-resistant character to the R1, R2, R3 and R4 generations was then investigated, and three plant lines which had an increased percentage of Al-resistant plants till the fourth generation of self-pollination, were identified. One was from a callus maintained on an Al-free medium. The efficiency of in vitro selection pressures was therefore debated.[PUBLICATION ABSTRACT]

A transgenic reconstruction experiment has been performed to determine the feasibility of male gametophytic selection to enhance transmission of genes to the next sporophytic generation. For tobacco pollen from a transgenic plant containing a single hygromycin-resistance (hygromycin phosphotransferase, hpt-) gene under control of the dc3 promoter, which is active in both sporophytic and gametophytic tissues, 3 days of in vitro maturation in hygromycin-containing medium was sufficient to result in a 50% reduction of germinating pollen, as expected for meiotic segregation of a single locus insert. Pollination of wild-type plants with the selected pollen yielded 100% transgenic offspring, as determined by the activity of the linked kanamycin-resistance gene-present within the same transferred T-DNA borders--under control of the nos promoter. This is direct proof that selection acting on male gametophytes can be a means to alter the frequency of genes in the progeny

Effectiveness of in vitro selection for agronomic characters was studied by finding correlation coefficients between in vitro and in vivo performance of 22 potato genotypes. Evaluation was performed under eight in vitro and two in vivo conditions. Genotypic differences were highly significant for various characters under all in vitro and in vivo conditions. Error mean squares were much lower in in vitro experiments than in in vivo experiments. In vitro selection was found to be highly effective for tuber colour, stem pigment and number of eyes, and moderately effective for average tuber weight, plant vigour and foliage senescence under specific conditions. The results also indicated the possibility of in vitro selection for heat tolerance. For tuber yield and number of tubers, effectiveness of in vitro selection was low to very low, because differences in phenotypic expressions of genotypes were much enlarged under in vitro conditions compared to under in vivo conditions. Correlation coefficients for certain characters were better when in vitro conditions were closer to in vivo conditions. To improve the efficiency of in vitro selection, there is perhaps a need to simulate and identify the in vitro conditions under which genotypic differences are reflected in phenotypes more realistically as they are expressed under in vivo conditions.[PUBLICATION ABSTRACT]

Microcuttings of three strains of 'McIntosh' apple that varied in their growth habit under field conditions were tested for their tolerance to three cytokinins (BA, kinetin, and 2iP) and one cytokinin-like compound (TDZ) in vitro. The three strains were 'McIntosh' (standard), 'Macspur' (intermediate) and 'Wijcik' (dwarf). For each cytokinin 'Wijcik' was more tolerant of high cytokinin levels than either 'Macspur', which showed an intermediate tolerance or 'McIntosh' which did not tolerate (died) high levels of cytokinins. These results show that 'Wijcik' exhibits multiple cytokinin tolerance in vitro. The relationship between growth habit and BA tolerance has been reported earlier. The present study demonstrates that cytokinins can be used to differentiate among growth types in apple.