Agrobacterium tumefaciens-mediated creeping bentgrass (Agrostis stolonifera L.) transformation using phosphinothricin selection results in a high frequency of single-copy transgene integration.

Genetic transformation of creeping bentgrass mediated by Agrobacterium tumefaciens has been achieved. Embryogenic callus initiated from seeds (cv. Penn-A-4) was infected with an A. tumefaciens strain (LBA4404) harboring a super-binary vector that contained an herbicide-resistant bar gene driven either by the CaMV 35S promoter or a rice ubiquitin promoter. Plants were regenerated from 219 independent transformation events. The overall stable transformation efficiency ranged from 18% to 45%. Southern blot and genetic analysis confirmed transgene integration in the creeping bentgrass genome and normal transmission and stable expression of the transgene in the T1 generation. All independent transformation events carried one to three copies of the transgene, and a majority (60-65%) contained only a single copy of the foreign gene with no apparent rearrangements. We report here the successful use of Agrobacterium for the large-scale production of transgenic creeping bentgrass plants with a high frequency of a single-copy transgene insertion that exhibit stable inheritance patterns.

Transformation of three genera of orchid using the bar gene as a selectable marker.

Protocorms or protocorm-like bodies (PLBs) of three orchid genera, Brassia, Cattleya, and Doritaenopsis, were genetically transformed via microprojectile bombardment using the bar gene from Streptomyces hygroscopicus. Selection for transformed cells was accomplished using bialaphos. PLBs that proliferated on selection medium containing 1 mg/l bialaphos were minced and transferred to selection medium containing 3 mg/l of the herbicide. This selection was repeated twice at 2-month intervals. Putatively transformed plantlets were regenerated in the absence of bialaphos for Doritaenopsis, or on regeneration medium containing 3 mg/l bialaphos for Brassia and Cattleya. Presence of the bar gene in the transformed plants was confirmed by polymerase chain reaction and Southern blot analysis. Transcription of bar was assessed by northern blot analysis. Plantlets of all three genera exhibited functional expression of bar as determined by assaying for resistance to bialaphos applied directly to leaves.

The structural, biochemical, and genetic characterization of a new radiation-induced, variegated leaf mutant of soybean [Glycine max (L.) Merr].

A variegated leaf mutant in soybean [Glycine max (L.) Merr.] has been identified and characterized. E25-10 was derived by exposure of seeds of the "Williams' 82" cultivar to gamma-radiation. In this mutant, yellow leaf sectors contain defective chloroplasts, in which the thylakoid membranes are presented as long, parallel structures with little or no overlap. No starch grains have been detected in the mutant chloroplasts. Small vesicles and plastoglobuli can be found within the defective chloroplasts. Genetic studies revealed that a single nuclear-encoded gene is responsible for the mutation in E25-10. The total chlorophyll content is reduced in yellow leaf tissue by 70-80%. However, the chlorophyll a/b ratio is not altered. The absorbance spectrum of pigments in the mutant leaf tissue differed from that of the green extracts in the range of 400-500 nm. This reduction in total chlorophyll and the change in the absorbance spectrum pattern in the yellow tissue is related to a loss of certain photosynthetic complexes. Green gel analysis revealed that four major pigment-protein complexes (CP1, LHCP1, LHCP2, and CPa) of the thylakoid membranes were absent in the E25-10 mutant. Lithium dodecyl sulphate polyacrylamide gel analysis showed that at least 5-6 polypeptides (51, 44, 25, 15, 13, and 12 kDa) were missing in the thylakoid membranes of chloroplasts from the yellow tissue. Changes in chloroplast- and nuclear-encoded gene message levels were detected. The psaA transcripts which code for the P700 apoprotein in PSI were reduced in chloroplasts from the E25-10 mutant yellow tissue. The levels of the large subunit of ribulose bisphosphate carboxylase (rbcL) and light harvest complex protein (LHCP) of PSII mRNA appeared to be reduced slightly in the mutant plants. However, a much more significant reduction in the 16S rRNA and the small subunit of ribulose bisphosphate carboxylase (rbcS) expression was detected in the yellow leaf sectors. Our results suggest that the possible lesion in E25-10 is located in the photosystem I even though fewer grana were observed in the defective chloroplasts.

A subtype of the metabotropic glutamate receptor family in the olfactory system of Atlantic salmon.

A plasma membrane rich fraction was prepared from olfactory rosettes of Atlantic salmon and used to study binding of L-glutamic acid and activation of phospholipase C (PLC). Glutamate binding was saturable, high affinity, and inhibited by aspartic acid and taurocholate but not by alanine and lysine. Binding of glutamate was potently inhibited by various ligands for rat brain metabotropic glutamate receptors (mGluR) and also by kainate and N-methyl-D-aspartate. Glutamate stimulated phosphatidylinositol 4,5-bisphosphate breakdown consistent with G protein-dependent activation of PLC. Northern blot analyses demonstrated the presence of olfactory rosette RNA that hybridizes with cDNA probes for mGluR1 and mGluR4 under low stringency conditions. The results indicate the salmon olfactory system includes a subtype of the metabotropic glutamate receptor family.

Unstable expression of a soybean gene during seed coat development.

The R gene of soybean is involved in anthocyanin synthesis in the seed coat, and its r-m allele conditions a variegated distribution of black spots and/or concentric rings of pigment superimposed on an otherwise brown seed coat. We describe an unusual feature of r-m that causes expression at the R locus to switch between active and inactive phases both somatically and germinally. Non-heritable somatic changes of the allele produce single plants containing mixtures of seed with different coat colors (black+striped or brown+striped). Heritable changes of the r-m allele are manifested in progeny plants which produce all black seed or all brown seed. Surprisingly, subsequent generations from revertant sublines show continued instability of the allele such that brown revertants (r*/r*) or homozygous black seed revertants (R*/R*) can give rise to striped or striped+black-seeded plants. Thus, the revertants produced by the r-m allele are not stable but interconvert between all three forms (R*, r*, and r-m) at detectable frequencies. Mutability of the r-m allele in a different genetic background has also been found after inter-crossing various soybean genotypes.

Regulation of Cat1 gene expression in the scutellum of maize during early sporophytic development.

A regulatory element has been identified in maize that appears to exert an effect specifically on Cat1 gene expression in the scutellum of maize during early sporophytic development. Cat1 encodes CAT-1 catalase, one of two forms of catalase expressed in the scutellum during this developmental time period. Density-labeling experiments indicate that the regulatory element influences the overall levels of CAT-1 protein synthesis in the scutellum but has no effect on CAT-2 protein synthesis. Immunoprecipitation experiments of in vitro translation products suggest that this element has an effect on the level of translatable Cat1 mRNA associated with the scutellar polysomes. The element exhibits additive inheritance and is tissue and time specific in its action. This element, therefore, meets all the criteria of a regulatory gene and has been designated Car2. The element acts to regulate the temporal expression of the Cat1 structural locus in maize.

Analysis of variants affecting the catalase developmental program in maize scutellum.

The catalase of maize scutella is coded for by two loci, Cat1 and Cat2, which are differentially expressed in this tissue during early seedling growth. Two variant lines have been previously identified in which the developmental program for the expression of the Cat2 structural gene in the scutellum has been altered. Line R6-67 exhibits higher than normal levels of CAT-2 catalase in this tissue after four days of postgerminative growth. This phenotype is controlled by a temporal regulatory gene designated Car1. Line A16 exhibits a CAT-2 null phenotype. Further analysis of Car1 verifies the initial indication that it is trans-acting and exhibits strict tissue (scutellum) specificity. A screen of other available inbred lines uncovered eight additional catalase high-activity lines. All eight lines exhibit significantly higher than normal levels of CAT-2 protein. Two of these lines have been shown to be regulated by Car1 as in R6-67. Another line (A338) uncovered during the screen exhibits a null phenotype for CAT-2 protein and resembles A16. Catalase activity levels are low in the scutellum and no CAT-2 CRM (cross-reacting material) is present in the tissues of this line. Also, unlike most maize lines, CAT-2 cannot be induced in the leaf tissue of A338 upon exposure to light. Finally, a single line (A337), demonstrating a novel catalase developmental program, was identified.

Purification and Partial Characterization of a Genetically-Defined Superoxide Dismutase (SOD-1) Associated with Maize Chloroplasts.

The chloroplast-associated form of superoxide dismutase from maize (Zea mays L.) (SOD-1) has been purified by a stepwise procedure consisting of (NH(4))(2)SO(4) fractionation, G-100 Sephadex gel filtration, DEAE-Sephacel chromatography, and hydroxylapatite chromatography. This procedure resulted in a single band on sodium dodecyl sulfate-polyacrylamide gels indicating that the preparation is homogeneous. The holoenzyme molecular weight was estimated at 31,000 to 33,000 by gel filtration. The subunit molecular weight of this dimeric protein was estimated at 14,500 on sodium dodecyl sulfate-polyacrylamide gels. Studies involving amino acid composition analysis, immunological cross-reactivity, in vitro subunit hybridizations, and H(2)O(2) sensitivity indicate that SOD-1 differs significantly from SOD-2 and SOD-4, the other cupro-zinc forms of SOD from maize. The possible physiological role of SOD-1 within the chloroplast is discussed.