FLP recombinase-mediated site-specific recombination in rice.

The feasibility of using the FLP/FRT site-specific recombination system in rice for genome engineering was evaluated. Transgenic rice plants expressing the FLP recombinase were crossed with plants harbouring the kanamycin resistance gene (neomycin phosphotransferase II, nptII) flanked by FRT sites, which also served to separate the corn ubiquitin promoter from a promoterless gusA. Hybrid progeny were tested for excision of the nptII gene and the positioning of the ubiquitin promoter proximal to gusA. While the hybrid progeny from various crosses exhibited beta-glucuronidase (GUS) expression, the progeny of selfed parental rice plants did not show detectable GUS activity. Despite the variable GUS expression and incomplete recombination displayed in hybrids from some crosses, uniform GUS staining and complete recombination were observed in hybrids from other crosses. The recombined locus was shown to be stably inherited by the progeny. These data demonstrate the operation of FLP recombinase in catalysing excisional DNA recombination in rice, and confirm that the FLP/FRT recombination system functions effectively in the cereal crop rice. Transgenic rice lines expressing active FLP recombinase generated in this study provide foundational stock material, thus facilitating the future application and development of the FLP/FRT system in rice genetic improvement.

Turf Grasses.

A reliable and efficient genetic transformation protocol for various turfgrass species and elite cultivars has been achieved using Agrobacterium tumefaciens. We describe a general protocol for the establishment of embryogenic cell cultures, Agrobacterium tumefaciens-mediated transformation, selection, and regeneration of transgenic turfgrass plants. Embryogenic callus is initiated from mature seeds, maintained by visual selection, and infected with an Agrobacterium tumefaciens strain (LBA4404) that contains either an herbicide-resistant bar gene or an antibiotic-resistant hyg gene driven either by a rice ubiquitin or CaMV35S promoter. Stable transformation efficiencies up to 43.3% were achieved. Southern blot and genetic analysis was used to confirm transgene integration in the turfgrass genomes and normal transmission and stable expression of the transgene in the T1 generation. We demonstrate herein that five elite cultivars of bentgrass can be genetically transformed using this single tissue culture media regime. Additionally, we report the successful Agrobacterium-mediated transformation of an elite tall fescue variety using minor variations in the same transformation protocol.

RTS, a rice anther-specific gene is required for male fertility and its promoter sequence directs tissue-specific gene expression in different plant species.

A tapetum-specific gene, RTS, has been isolated by differential screening of a cDNA library from rice panicles. RTS is a unique gene in the rice genome. RNA blot analysis and in situ hybridization indicates that this gene is predominantly expressed in the anther's tapetum during meiosis and disappears before anthesis. RTS has no introns and encodes a putative polypeptide of 94 amino acids with a hydrophobic N-terminal region. The nucleotide and deduced amino acid sequence of the gene do not show significant homology to any known sequences. However, a sequence in the promoter region, GAATTTGTTA, differs only by one or two nucleotides from one of the conserved motifs in the promoter region of two pollen-specific genes of tomato. Several other sequence motifs found in other anther-specific promoters were also identified in the promoter of the RTS gene. Transgenic and antisense RNA approaches revealed that RTS gene is required for male fertility in rice. The promoter region of RTS, when fused to the Bacillus amyloliquefaciens ribonuclease gene, barnase, or the antisense of the RTS gene, is able to drive tissue-specific expression of both genes in rice, creeping bentgrass (Agrostis stolonifera L.) and Arabidopsis, conferring male sterility to the transgenic plants. Light and near-infrared confocal microscopy of cross-sections through developing flowers of male-sterile transgenics shows that tissue-specific expression of barnase or the antisense RTS genes interrupts tapetal development, resulting in deformed non-viable pollen. These results demonstrate a critical role of the RTS gene in pollen development in rice and the versatile application of the RTS gene promoter in directing anther-specific gene expression in both monocotyledonous and dicotyledonous plants, pointing to a potential for exploiting this gene and its promoter for engineering male sterility for hybrid production of various plant species.

Use of beta-glucuronidase reporter gene for gene expression analysis in turfgrasses.

The beta-glucuronidase (GUS) gene has been successfully used as a reporter gene in innumerable number of plant species. The functional GUS gene produces blue coloration in plants upon integration into the plant genome. Because of the ease it provides to analyze the gene expression (as no expensive equipment is needed), GUS gene is surely plant biotechnologist's first choice as a reporter gene. The turfgrass family contains the world's most economically important horticultural crops. There is a world-wide drive for genetic modification of grasses due to its huge economic importance. GUS gene can be transiently or stably expressed in grasses for the purpose of promoter analysis and to study tissue-specific and developmental gene expression. This paper summarizes the use of GUS gene for transient and stable expression studies in various turfgrass species.

Promoter analysis in transient assays using a GUS reporter gene construct in creeping bentgrass (Agrostis palustris).

Transient expression profiles for several chimeric beta-glucuronidase (GUS) gene constructs were determined in tissues (young leaves, mature leaves and roots) of creeping bentgrass (Agrostis palustris, cv. Penn A4) following microprojectile bombardment. The constructs analyzed consisted of the uidA (GUS) reporter gene driven by four different promoters (ubiquitin 3-potato, ubiquitin corn, ubiquitin rice and CaMV 35S). The total number of GUS hits (or transient expression units; TEUs) were determined manually under a dissecting scope after histochemical staining for GUS. Results suggest that the ubiquitin rice promoter is most active in cells of turfgrass, regardless of the developmental stage or tissue-type. The ubiquitin corn promoter was the next best. Of the four promoter used, except for ubiquitin 3-potato, reporter gene activity was dramatically higher in mature leaves compared to young leaves. The relative efficiency of each promoter was about the same in roots and leaves. We have also analyzed uidA (GUS) reporter gene activity following microprojectile bombardment in transient expression assays with callus from two cultivars (Providence or Penn A4) of creeping bentgrass. Differences in the frequency of GUS positive hits were observed between cultivars up to 72 hours post-bombardment. However, this difference between cultivars disappeared after 72 hours post-bombardment. This information describing promoter functionality in bentgrass will be important when designing gene constructs for trait modification and when choosing appropriate cultivars for improvement through gene transfer experiments. This is the first in depth report on organ-specific and developmental gene expression profiles for transgenes in a turfgrass species.

Transient reporter gene (GUS) expression in creeping bentgrass (Agrostis palustris) is affected by in vivo nucleolytic activity.

Leaf and callus tissues of a creeping bentgrass cultivar (Penn A4) had high nuclease activities that degraded exogenously added plasmid DNA. When callus tissue was incubated for 24 h with heparin, spermidine, aurintricarboxylic acid or polyethylene glycol, only heparin and spermidine were effective as in vitro nuclease inhibitors, protecting exogenously added plasmid DNA from degradation. When beta-glucuronidase (GUS) reporter gene activity was evaluated in heparin-treated (0.6%), 14-month old callus following microprojectile bombardment, GUS activity increased 1000-fold compared to equivalent aged untreated Penn A4 callus. Similar enhancement from heparin pretreatment (0.6% or 1.2%) was not observed in 6-month old callus. This is likely due to much higher activities of nuclease in the younger callus.

Morphological, Pathogenic, and Genetic Comparisons of Colletotrichum graminicola Isolates from Poaceae.

Isolates of Colletotrichum graminicola from annual blue grass and creeping bent grass were investigated for their morphological characteristics, host specificity, and genetic relatedness. One isolate from maize and one from sorghum (C. sublineolum) were included for comparison. Recently isolated cultures of C. graminicola from annual blue grass were readily distinguished from those isolated from creeping bent grass on the basis of pigmentation. Differences in appressoria size and shape were found only between the turf grass isolates and those from maize and sorghum. Spore length varied significantly between host groups. Differences in host range and virulence were also apparent. In general, isolates from creeping bent grass incited disease on both creeping bent grass and annual blue grass, while those from annual blue grass essentially were limited to host. Random amplified polymorphic DNA (RAPD) marker analysis of C. graminicola isolates from turf grass revealed that a high degree of genetic similarity exists among isolates recovered from the same host, but exceptions were found. Therefore, an absolute distinction between isolates recovered from two turf grass hosts could not be made based on RAPD markers.