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.

Mesophyll-specific, light and metabolic regulation of the C4 PPCZm1 promoter in transgenic maize.

To play an essential role in C4 photosynthesis, the maize C4 phosphoenolpyruvate carboxylase gene (PPCZm1) acquired many new expression features, such as leaf specificity, mesophyll specificity, light inducibility and high activity, that distinguish the unique C4 PPC from numerous non-C4 PPC genes in maize. We present here the first investigation of the developmental, cell-specific, light and metabolic regulation of the homologous C4 PPCZm1 promoter in stable transgenic maize plants. We demonstrate that the 1.7 kb of the 5'-flanking region of the PPCZm1 gene is sufficient to direct the C4-specific expression patterns of beta-glucuronidase (GUS) activity, as a reporter, in stable transformed maize plants. In light-grown shoots, GUS expression was strongest in all developing and mature mesophyll cells in the leaf, collar and sheath. GUS activity was also detected in mesophyll cells in the outer husks of ear shoots and in the outer glumes of staminate spikelets. We did not observe histological localization of GUS activity in light- or dark-grown callus, roots, silk, developing or mature kernels, the shoot apex, prop roots, or pollen. In addition, we used the stable expressing transformants to conduct and quantify physiological induction studies. Our results indicate that the expression of the C4 PPCZm1-GUS fusion gene is mesophyll-specific and influenced by development, light, glucose, acetate and chloroplast biogenesis in transgenic maize plants. These studies suggest that the adoption of DNA regulatory elements for C4-specific gene expression is a crucial step in C4 gene evolution.

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.

Organelle isolation by magnetic immunoabsorption.

Currently, most organelle isolation procedures rely on physical parameters and centrifugation for separation. Here, we report the rapid and gentle isolation of a variety of organelles by immunolabeling whole cell lysates with organelle-specific antibodies and streptavidin magnetic particles followed by separation in a magnetic field. Using magnetic immunoabsorption, we have been able to specifically label mouse metaphase chromosomes and a variety of plant organelles, including: amyloplasts, choroplasts and nuclei from whole cell lysates of various plant tissues. We find that the distinct magnetic properties, surface characteristics and mean diameter-size ranges of different particle preparations significantly influence their specific utility for organelle isolations. By using an internal-field magnetic separation device, we have developed a method for quantitative recovery of labeled organelles in microarrays and tested a variety of antibodies to chloroplast outer envelope proteins for their ability to immune-isolate chloroplasts.

Transformation of Maize Cells and Regeneration of Fertile Transgenic Plants.

A reproducible system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed with the bacterial gene bar using microprojectile bombardment. Transformed calli were selected from the suspension cultures using the herbicide bialaphos. Integration of bar and activity of the enzyme phosphinothricin acetyltransferase (PAT) encoded by bar were confirmed in all bialaphos-resistant callus lines. Fertile transformed maize plants (R0) were regenerated, and of 53 progeny (R1) tested, 29 had PAT activity. All PAT-positive progeny analyzed contained bar. Localized application of herbicide to leaves of bar-transformed R0 and R1 plants resulted in no necrosis, confirming functional activity of PAT in the transgenic plants. Cotransformation experiments were performed using a mixture of two plasmids, one encoding PAT and one containing the nonselected gene encoding [beta]-glucuronidase. R0 plants regenerated from co-transformed callus expressed both genes. These results describe and confirm the development of a system for introduction of DNA into maize.

Light regulation of plant gene expression by an upstream enhancer-like element.

Light regulates many varied physiological and developmental phenomena during plant growth and differentiation, including the formation of a photosynthetically competent chloroplast from a proplastid. The expression of ribulose 1,5-bisphosphate carboxylase small subunit (rbcS) genes is regulated by light in a development- and tissue-specific manner2,3. In some plant species, phytochrome has been demonstrated to mediate this response, and photoregulation of rbcS expression occurs at least in part at the level of transcription. We have shown previously that a 5'-noncoding fragment (4-973 base pairs (bp) upstream of the messenger RNA cap site) of the pea rbcS ss3.6 gene contains all of the nucleotide sequence information necessary to direct the photoregulated expression of a bacterial chloramphenicol acetyltransferase (cat) gene in tobacco. Consistent with these findings, Morelli et al.11 have shown by deletion analysis of a second rbcS gene promoter, that the sequences required for photoregulated expression of rbcS E9 reside within the 5'-noncoding region. They identified an upstream region of approximately 700 bp needed for maximum transcription but not light-dark regulation, and a region from -35 to -2 bp which included the TATA box and contained the necessary information for light responsiveness. We now demonstrate that regulatory sequences 5' distal to the rbcS ss3.6 TATA box and transcriptional start site not only contain the information necessary for maximum expression, but also confer photoregulation. These upstream regulatory sequences function independently of orientation when fused to their homologous promoter or a heterologous promoter.

Molecular characterization and genetic mapping of two clusters of genes encoding chlorophyll a/b-binding proteins in Lycopersicon esculentum (tomato).

We have constructed a tomato genomic library in the gamma Charon 4 phage vector. The library was screened with a pea cDNA probe encoding a chlorophyll a/b-binding protein (CAB), and several recombinant phages containing tomato CAB genes were isolated and characterized by restriction mapping, heteroduplex analysis and nucleotide sequencing. Two phages with overlapping segments of the tomato genome contain a total of four CAB genes, all arranged in tandem. A third phase contains three CAB genes, two arranged in tandem and one in opposite orientation, and an additional, truncated CAB gene. Genetic mapping experiments showed that the four CAb genes on the first two phages belong to a locus, previously designated Cab-1, on chromosome 2. The CAB genes from the third phage belong to the Cab-3 locus on chromosome 3. Complete sequence determination of two CAB genes, one from each locus, and additional sequence determination of about 50% of each of the other five CAB genes showed that each gene within a CAB locus is more similar to other CAB genes in the same locus than it is to the CAB genes from the second locus. Furthermore, the polypeptides encoded by Cab-1 genes diverge significantly from those encoded by Cab-3 genes in the domains of transit peptide and the N terminus of the mature polypeptide but are essentially identical in the rest of the sequence.

Targeting of a foreign protein to chloroplasts by fusion to the transit peptide from the small subunit of ribulose 1,5-bisphosphate carboxylase.

Chimaeric genes can be constructed which fuse the transit peptide of a small subunit of the chloroplast-located ribulose 1,5-bisphosphate carboxylase with a bacterial protein. The fusion protein is translocated into chloroplasts and cleaved in a similar way to the small subunit polypeptide precursor.

Absence of CeCl3-detectable peroxisomal glycolate-oxidase activity in developing raphide crystal idioblasts in leaves of Psychotria punctata Vatke and roots of Yucca torreyi L.

Three peroxisomal enzymes, glycolate oxidase, urate oxidase and catalase were localized cytochemically in Psychotria punctata (Rubiaceae) leaves and Yucca torreyi (Agavaceae) seedling root tips, both of which contain developing and mature calcium-oxalate raphide crystal idioblasts. Glycolate-oxidase (EC 1.1.3.1) and catalase (EC 1.11.1.6) activities were present within leaftype peroxisomes in nonidioblastic mesophyll cells in Psychotria leaves, while urate-oxidase (EC 1.7.3.3) activity could not be conclusively demonstrated in these organelles. Unspecialized peroxisomes in cortical parenchyma of Yucca roots exhibited activities of all three enzymes. Reactionproduct deposits attributable to glycolate-oxidase activity were never observed in peroxisomes of any developing or mature crystal idioblasts of Psychotria or Yucca. Catalase localization indicates that idioblast microbodies are functional peroxisomes. The apparent absence of glycolate oxidase in crystal idioblasts of Psychotria and Yucca casts serious doubt that pathways involving this enzyme are operational in the synthesis of the oxalic acid precipitated as calcium-oxalate crystals in these cells.

Biogenesis and cytochemistry of unspecialized peroxisomes in root cortical cells of Yucca torreyi L.

Microbodies containing bipyramidal crystalline nucleoid inclusions occur within every cortical cell in roots of Yucca torreyi. Reaction product deposition attributable to catalase, glycolate oxidase, and urate oxidase activities are cytochemically localized to Yucca root microbodies and classifies them as unspecialized peroxisomes on the basis of their enzyme complement and tissue origin. Crystalline nucleoids do not stain for glycolate or urate oxidase activities, appearing as negatively-stained inclusions, but are apparently reactive for catalase activity. Development of unspecialized peroxisomes in Yucca roots is consistent with all evidence for glyoxysome and leaf-type peroxisome biogenesis from ER. Dilated ends of ER cisternae accumulate cytochemically detectable glycolate oxidase activity. After considerable dilation, paracrystalline precursors to nucleoids form within the bulge, and the inclusion enlarges to comprise the majority of peroxisomal volume. Peroxisomes that are not attached to ER are observed with high voltage electron microscopy and in serial thin sections, implying that eventually the budding peroxisomes are vesiculated. The functions of these unspecialized peroxisomes are suggested based upon cytochemical detection of their partial enzyme complement and their spatial and developmental timing relationships within developing Yucca root cortical parenchyma cells.

Increased nuclear DNA content in raphide crystal idioblasts during development in Vanilla planifolia L. (Orchidaceae).

Longitudinal files of raphide crystal idioblasts form within the cortical meristematic region of Vanilla planifolia aerial roots. Cell and nuclear enlargement occur gradually throughout idioblast development and nuclear diameter approximates idioblast maturity. Cytophotometric determination of nuclear DNA (Feulgen) contents, measured by the two-wavelength method, revealed that all cortical parenchyma cells are diploid (2C = 6.3 pg), whereas all crystal idioblast nuclei are endopolyploid. Idioblast nuclear DNA content ranged from 4C to 32C (106 pg) and averaged 5.9 times that of parenchyma telophase nuclei. Frequency distribution of individual DNA content measurements depicts multiple genomes (increasing with geometric periodicity) to the 8C level, followed by less strict DNA replication within the crystal idioblast genome. The largest nuclei had the highest DNA content. Endomitotic stages of preprophasic heterochromatic dispersion (Z phase) and partial prophasic chromosomal coiling are observed with light and electron microscopy. DNA content values above the 8C level do not fit the geometrical order which is found if the total genome is replicated during each endo-cycle, a result indicating differential DNA replication. Chromocenter counts substantiate the occurrence of endomitosis to the 8C level and suggest heterochromatin underreplication in higher endopolyploid idioblast nuclei. Possible relationships between observed cytological events of idioblast development and nuclear condition are discussed.