HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Figures Only Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sandhu, S. Articles by Blount, A. R. Search for Related Content PubMed Articles by Sandhu, S. Articles by Blount, A. R. Agricola Articles by Sandhu, S. Articles by Blount, A. R. Related Collections Forage Management Turfgrass Management Turfgrass Pesticides

GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Apomictic Bahiagrass Expressing the bar Gene Is Highly Resistant to Glufosinate under Field Conditions

^a Agronomy Dep., Plant Molecular and Cellular Biology Program, Genetics Institute, Univ. of Florida-IFAS, Gainesville, FL 32611
^b NFREC, Agronomy Dep., Univ. of Florida-IFAS, Marianna, FL

^* Corresponding author (faltpeter{at}ifas.ufl.edu).

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Bahiagrass (Paspalum notatum Flugge) is one of the most important low-input turf and forage grasses in the southeastern USA and in other subtropical regions. Its open growth habit, however, facilitates weed encroachment and its low tolerance to commercially available herbicides complicates weed management. Genetic transformation protocols were recently developed for bahiagrass that allow now, among other approaches, the introduction of herbicide resistance genes. Integration of the bar gene expression cassette into the genomic DNA of the apomictic bahiagrass cultivar Argentine was confirmed by Southern blot analysis. Stable expression of the bar gene was detected by an immunochromatographic assay in primary transgenic lines and the seed-derived progeny plants. Several independent transgenic lines showed no growth inhibition, chlorosis, or necrosis following a spray application of 1.0% glufosinate ammonium [2-amino-4-(hydroxymethylphosphinyl)butanoic acid] under greenhouse conditions. The selected transgenic plants did not differ morphologically from wild-type plants and produced viable seeds in the greenhouse and field. All weeds that coestablished during the field experiment, as well as wild-type bahiagrass, displayed full leaf necrosis after application of 0.3% glufosinate ammonium. Transgenic bahiagrass plants were resistant to a field application of 0.6% glufosinate ammonium, which is twice the recommended rate for weed control, without any symptoms of phytotoxicity.

Abbreviations: PAT, phosphinothricin acetyltransferase

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
BAHIAGRASS (Paspalum notatum Flugge) is an important turf and forage grass in the southeastern USA and in subtropical regions around the world. Commercially important cytotypes include sexually reproducing, cross-pollinating diploids (2n = 2x = 20) like ‘Pensacola’ or ‘Tifton 9’ and apomictic, tetraploid (2n = 4x = 40) genotypes like ‘Argentine’ (Gates et al., 2004). Genetic improvement of tetraploid bahiagrass cultivars has been compromised by the absence of genetic recombination in apomictic cytotypes. Genetic manipulation through cellular and molecular techniques will overcome this hurdle. The obligate apomict Argentine (Burton, 1948) represents the preferred target for genetic transformation to incorporate herbicide resistance. Its apomictic mode of reproduction will allow generation of uniform transgenic seed progeny with a reduced risk of unintended gene dispersal by pollen. Bahiagrass is drought and heat tolerant and resists most insects and diseases. Its open growth habit, however, supports weed encroachment and its very low tolerance to commercially available herbicides make weed management a difficult task. Hence, development of herbicide-resistant Argentine bahiagrass will reduce weed management problems for this low-input grass.

Glufosinate ammonium is a postemergence herbicide widely used for broad-spectrum weed control. The short half-life (7 d) of glufosinate ammonium limits soil residual activity and makes it environmentally benign. Commercially available glufosinate ammonium resistant crops include cotton (Gossypium hirsutum L.), corn (Zea mays L.), and canola (Brassica napus L. var. napus) (Castle et al., 2006). Transgenic glufosinate ammonium herbicide resistant turf and forage grasses have been reported from several species including creeping bentgrass (Agrostis palustris Huds.; Hartman et al., 1994; Luo et al., 2004), bermudagrass [Cynodon dactylon (L.) Pers.; Goldman et al., 2004; Li et al., 2005] and Kentucky bluegrass (Poa pratensis L.; Gao et al., 2006). Transgenic plants expressing the bar gene of the non-commercial apomictic genotype ‘Tifton 7’ and the diploid bahiagrass Pensacola have been recently reported (Smith et al., 2002; Gondo et al., 2005). Herbicide resistance was not investigated in these transgenic bahiagrass lines, however, other than regeneration of plants from bialaphos-containing culture medium (Gondo et al., 2005) or leaf painting of primary transgenic plants with 0.4% of glufosinate ammonium (Smith et al., 2002).

This is the first study of bar transgene expression in apomictic progeny of the commercially important bahiagrass cultivar Argentine and identification of transgenic lines with high-level herbicide resistance under both greenhouse and field conditions.

	MATERIALS AND METHODS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Callus Induction
Embryogenic callus was induced from germinating seedlings as described by Altpeter and James (2005). The callus induction medium (CIM) consisted of Murishige & Skoog (MS) salts (Murashige and Skoog, 1962), 30 g L^–1 sucrose, 1.1 mg L^–1 6-benzylaminopurine (BAP), 3 mg L^–1 3,6-dichloro-2-methoxy benzoic acid (dicamba), and 6 g L^–1 agarose (Sigma, St. Louis, MO), supplemented with filter-sterilized MS vitamins (Murashige and Skoog, 1962), which were added after the medium was autoclaved for 20 min. Calli were kept in darkness at a temperature of 28°C and subcultured to fresh CIM biweekly.

Gene Expression Cassettes
Plasmid p35S-int-nptII contains the selectable marker gene nptII gene (Bevan, 1984) under the transcriptional control of the constitutive promoter CaMV 35S promoter (Odell et al., 1985) with the HSP70 intron (Rochester et al., 1986) and CaMV 35S 3'UTR (Dixon et al., 1986). The pJFbar plasmid contains the bar gene (encoding phosphinothricin acetyltransferase) (Thompson et al., 1987), under transcriptional control of the constitutive maize ubiquitin promoter, first intron (Christensen and Quail, 1996) and CaMV 35S 3'UTR (Dixon et al., 1986; Fig. 1 ).

View larger version (19K): [in this window] [in a new window]   Figure 1. Diagrammatic representation of the bar gene expression cassette used for genetic transformation of bahiagrass (Paspalum notatum Flugge).

Biolistic Transformation, Plant Regeneration, and Propagation
The nptII and bar gene expression cassettes were used in a 1:2 molar ratio and coprecipitated on 1.0-µm diameter gold particles as described by Altpeter and James (2005). Embryogenic calli were placed on CIM medium supplemented with 0.4 mol L^–1 sorbitol, for 4 to 6 h before gene transfer. The BioRad PDS-1000/He device (Sanford et al., 1990) was used for biolistic gene transfer at 7.584 MPa and 3733 Pa. The bombarded calli were transferred to fresh CIM medium following the gene transfer and kept in the dark for 10 d. Then they were transferred to low light conditions (30 µmol m^–2 s^–1), with 16/8-h (light/dark) photoperiod, at 28°C, on selection CIM medium containing 50 mg L^–1 of paromomycin. After 4 wk, the calli were subcultured on shoot regeneration medium, similar to CIM except containing 0.1 mg L^–1 BAP and no dicamba. These cultures were transferred to an illuminated (150 µmol m^–2 s^–1) incubator with a 16/8-h photoperiod at 28°C. To induce root formation, calli were transferred to hormone-free CIM medium. The regenerated plantlets were transplanted into Fafard 2 soil mix (Fafard, Apopka, FL) and acclimatized in growth chambers set at the conditions described above, and later moved to an air-conditioned greenhouse at 30/20°C (day/night) and natural photoperiod. Plants were fertilized biweekly with Miracle-Gro Lawn Food (Scotts Miracle-Gro Products, Marysville, OH) at the recommended rate. Visual observations of plant morphology were recorded. To evaluate the viability of seeds, the seed coat was removed with a scalpel to break the seed dormancy and seedlings were evaluated for transgene expression.

Glufosinate Application
Greenhouse Application
Ignite (18.1% glufosinate ammonium a.i., AgrEvo, Wilmington, DE) was used for herbicide applications. Thirty-two putative transgenic lines were sprayed with 0.2% glufosinate ammonium using a hand sprayer. Visual observations of plant injury or other symptoms were recorded 14 d after herbicide application. Eighteen of the above lines were further tested at three different concentrations of glufosinate ammonium. A completely randomized experimental design with two replications per line and three treatment concentrations, 0.2, 0.5, or 1.0% glufosinate ammonium, was used. One liter of a herbicide mixture containing eight drops of Tween 20 (Fisher Biotech, NJ) was used to treat 38 individual plants. Using a hand sprayer, each plant was dosed until the herbicide ran off. Visual observations for plant injury were scored 14 d after herbicide application. The degree of plant injury was scored on a 1 to 5 scale as follows: 1, plant with no injury; 2, two to three leaves per plant display necrosis at the leaf tip (<10% of the leaf area); 3, most leaves display 10 to 40% leaf necrosis; 4, most leaves display 40 to 80% leaf necrosis; 5, all leaves display 80 to 100% leaf necrosis.

Field Application
The USDA Animal and Plant Health Inspection Service (USDA-APHIS Permit 05-365-01r) allowed evaluation and seed production of transgenic herbicide-resistant lines under field conditions in a designated plot in Marianna, FL. Highly resistant transgenic lines 2, 3, and 33, along with the wild type, were vegetatively propagated in the greenhouse and transplanted into the field by the end of May 2006. Transgenic lines were transplanted in the center of 1-m diameter circular plots with wild-type plants located to the outside. There were four plants of each transgenic line per plot and each circular plot setup was replicated four times. Viable seeds were produced on wild-type and transgenic bahiagrass plants during July and August following a single N application of 30 kg ha^–1. Herbicide resistance of the above lines was tested by applying 0.3 and 0.6% glufosinate ammonium in two plots per treatment on 1 Sept. 2006. Applying 0.3% glufosinate ammonium is the recommended field rate for control of broadleaf and grassy weeds. The herbicide was applied until runoff using a hand sprayer. Visual observations were recorded 14 d after herbicide application.

Immunodetection of Phosphinothricin Acetyltransferase Using a Lateral-Flow Membrane Strip
An immunochromatographic detection kit that uses lateral-flow membrane strips (Quick Stix for LibertyLink/bar, Envirologix, Portland, ME) for the analysis of the bar gene product phosphinothricin acetyltransferase (PAT) was used in this study. Total protein was extracted with the buffer solution provided in the kit, and the protein concentration was estimated using the Bradford assay (Bradford, 1976). Protein extracts (50 µL) at a concentration of 1 g L^–1 were used for the assay. Protein extracts from nontransformed wild-type bahiagrass were used as negative controls. The lateral-flow membrane assay was performed on all lines that showed no injury after application of 0.2% glufosinate ammonium and on T₁ progeny from selected lines.

Southern Hybridization
Fresh leaf tissue (3 g) was used to extract genomic DNA by the CTAB method (Murray and Thompson, 1980). Genomic DNA (20 µg) digested with BglII was electrophoresed overnight at 20 V on a 1.0% agarose gel in 1x Tris–acetate–EDTA buffer (Sambrook and Russell, 2001) and then transferred to Hybond N membrane (Amersham Pharmacia Biotech, Piscataway, NJ) with 0.4 mol L^–1 NaOH (Sambrook and Russell, 2001). The full-length coding region of the bar gene, (0.56 kb) used as a probe was obtained by restriction digestion of pJFbar with MluI and XhoI followed by gel extraction (QIAquick gel extraction kit, Qiagen, Valencia, CA). Labeling of the probe with [³²P] deoxy cytidine 5'-triphosphate was done using the Prime-a-gene labeling system (Promega, Madison, WI). The membrane was prehybridized for 4 h at 65°C and then hybridized overnight in the hybridization buffer containing denatured salmon sperm DNA (1 g L^–1) and the labeled probe (Sambrook and Russell, 2001). The membrane was washed with 0.1X SSC buffer and 0.1% sodium dodecyl sulfate at 65°C before exposure to Kodak Biomax MS autoradiography film at –80°C for 30 h.

	RESULTS

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Regeneration of Transgenic Plants
Growth, regeneration of plants (Fig. 2a ), and root formation on paromomycin-containing culture medium allowed selection of 32 independent putative transgenic plants (Fig. 2b) from a total of 300 bombarded callus pieces and a total of 10 bombardments.

View larger version (92K): [in this window] [in a new window]   Figure 2. Generation and evaluation of herbicide-resistant bahiagrass: (a) regeneration of plants from embryogenic bahiagrass callus following gene transfer; (b) transgenic bahiagrass plants established in soil in the greenhouse; (c) response of nontransgenic, wild-type bahiagrass (WT) and transgenic bahiagrass line 4 or (d) line 24 expressing the bar gene, 14 d after application of 0.2, 0.5, or 1.0% glufosinate ammonium under greenhouse conditions; (e) seed-derived progeny plants of transgenic bahiagrass; (f) transgenic bahiagrass lines expressing the bar gene (center) surrounded by nontransgenic bahiagrass plants before and (g) 14 d after application of 0.6% glufosinate ammonium under field conditions; (h) immunodetection of bar-encoded phosphinothricin acetyltransferase (PAT) in T0 transgenic lines or (i) seed-derived progeny of transgenic plants by lateral flow membrane assay (Libertylink Stix test). Protein extracts with detectable levels of PAT display a PAT-specific band indicated by the arrow, whereas extracts from nontransgenic, wild-type (WT) bahiagrass show a separate band, common to all lines, which indicates the functionality of the lateral flow membrane strip.

Further testing of the level of herbicide tolerance was performed by applying 0.2, 0.5, and 1.0% glufosinate ammonium to 18 randomly selected herbicide-tolerant lines, as well as wild type, and one susceptible line (line 16). These lines showed differences in the response to applications of higher concentrations of glufosinate ammonium (0.5 and 1.0%; Table 1, Fig. 2c and 2d). Highly resistant lines including 2, 3, 4, 11, and 33 did not display any injury or stress symptoms even at the highest application rate of at 1.0% glufosinate ammonium (injury score = 1, Table 1; Fig. 2c). Lines 7, 10, 19, 28, and 52 showed no injury following an application of 0.2% glufosinate ammonium, and only mild or no injury at 0.5% glufosinate ammonium as well as minor injury symptoms at 1.0% glufosinate ammonium (score = 1.5–2, Table 1). Lines 6, 21, 22, 24, 32, 36, 38, and 39 displayed moderate leaf injury (scores between 2 and 3) at the lowest glufosinate ammonium concentration, but still survived applications of 0.5% or 1.0% glufosinate ammonium (Fig. 2d). Herbicide leaf injury in line 24 was moderate; however, this line displayed a growth inhibition following application of 0.5 or 1.0% of glufosinate ammonium. Wild-type bahiagrass showed 100% leaf necrosis at all glufosinate ammonium application levels.

Molecular Analysis of the Herbicide-Resistant Lines
Immunodetection of the bar gene product PAT was accomplished by a lateral-flow membrane assay (Quick Stix for Liberty Link, Envirologix; Fig. 2h and 2i). All bahiagrass lines that were resistant to 0.2% glufosinate ammonium application showed the PAT-specific band in the immunochromatographic assay (indicated with an arrow in Fig. 2h), in contrast to the wild-type and glufosinate ammonium susceptible lines 16 and 40, which did not display this band (data not shown). Stable bar expression in apomictic progeny plants was indicated by the presence of the PAT-specific band in the immunochromatographic assay (Fig. 2i). Southern blot analysis of the genomic DNA of herbicide-resistant bahiagrass lines confirmed the stable integration of the bar gene (Fig. 3 ). All lines showed different integration patterns, indicating that they were obtained from independent transformation events. Most transgenic lines showed complex bar gene integration patterns. An exception to this trend was line 7, with approximately two copies of the bar gene.

View larger version (89K): [in this window] [in a new window]   Figure 3. Southern blot analysis of transgenic bahiagrass. Genomic DNA (20 µg) from nontransgenic wild-type (WT) and transgenic bahiagrass lines (4–52) was digested with BglII, which cuts once in the expression cassette of the bar gene (Fig. 1). The full length coding region of the bar gene was used as a probe and labeled with 32P-deoxy cytidine 5'-triphosphate. Linearized pJFBar plasmid (20 pg) was used as a positive control (PC).

	DISCUSSION

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Gondo et al. (2005) have reported the production of a herbicide-resistant diploid bahiagrass cytotype (cv. Pensacola); however, regeneration of plants on bialaphos-containing culture medium was the only presented evidence of herbicide resistance. In contrast, the experiments described here focused on the production of transgenic bahiagrass with resistance to high concentrations of glufosinate ammonium. Application of glufosinate ammonium under greenhouse conditions at different concentrations (0.2, 0.5, or 1.0%) identified different levels of herbicide resistance in independent transgenic bahiagrass lines ranging from no to moderate injury at the highest (1.0%) glufosinate ammonium application level (Table 1). Wild-type bahiagrass showed 100% leaf necrosis at all glufosinate ammonium application levels. Glufosinate ammonium is an inhibitor of glutamine synthase, a key enzyme in the N assimilation pathway. Glutamine synthase inhibition leads to NH₃ accumulation, resulting in phytotoxicity. The bar gene encodes PAT, which inactivates glufosinate ammonium by acetylating its active amino group. Low-level herbicide resistance in some of the lines is probably a result of inadequate PAT levels, thus resulting in partial leaf necrosis. Glufosinate ammonium has also been shown to have an indirect effect on plant photosynthesis, which may be responsible for the growth inhibition observed in line 24 (Wild and Wendler, 1993). Representative lines from each resistance level were selected for Southern blot analysis. Multiple transgene copies have been associated with reduced expression levels, due to transgene silencing at the transcriptional or post-transcriptional level (Wang and Waterhouse, 2000). The complex transgene integration pattern of transgenic line 52 resulted in a similar, moderate herbicide resistance level as in line 7 that contained approximately two copies of the bar gene (Fig. 3, Table 1). Further, line 52 stably expressed the transgene in progeny seedlings (Fig. 2e), as detected by immunochromatography (Fig. 2i), despite its complex transgene integration pattern (Fig. 3). On the other hand, highly resistant lines 4 and 33 had fewer copies of the bar gene compared with the moderately resistant lines 28 and 52. The observed line-to-line variation could be due to the position of transgene insertion and may be influenced by the transcriptional activity of neighboring regions or by flanking endogenous regulatory sequences (Meyer, 2000). A common band of approximate 3 kb was observed in all transgenic lines, which could be due to the formation of concatemers. This rather complex transgene integration pattern was observed despite the use of minimal cassettes without vector backbone. In contrast, Fu et al. (2000) observed a simple integration pattern and lower copy numbers with minimal cassettes than plasmids. Consistent with our findings, however, Breitler et al. (2002) also described highly complex transgenic loci following the use of minimal cassettes. This suggests that the complexity of a transgenic locus depends on factors intrinsic to the plant and not to the transgene, as stated earlier by Agrawal et al. (2005).

All naturally occurring tetraploid bahiagrass genotypes are considered apomictic (Burton, 1948). The commercially important apomictic cultivar Argentine used in this study represents the preferred target for genetic transformation to incorporate herbicide resistance. Its apomictic mode of reproduction supports the generation of uniform transgenic seed progeny with a reduced likelihood of unintended gene dispersal by pollen.

Transgenic lines 2, 3, and 33 were established in the field under USDA-APHIS permit no. 05-365-01r and herbicide resistance was evaluated by application of 0.3 and 0.6% of glufosinate ammonium. The application of 0.3% glufosinate ammonium is the recommended application rate, and it resulted in 100% leaf necrosis of nontransgenic bahiagrass and all the annual and perennial weeds that coestablished during the field trial. Figure 2g shows the difference between the glufosinate ammonium resistant transgenic lines 2, 3, and 33 in the center and the wild-type bahiagrass plants surrounding them that display 100% necrosis. All of these transgenic bahiagrass lines had a normal phenotype and produced viable seeds under greenhouse and field conditions.

Bahiagrass seedlings easily succumb to weed pressure until they are 13 to 15 cm tall, and are very sensitive to phenoxy herbicides (Chambliss and Adjei, 2006). Bahiagrass pasture establishment is variable due to poor seedling vigor, slow germination, and sensitivity to soil moisture. Poor stands of bahiagrass encourage weed infestation, and reseeding a poorly established pasture of bahiagrass without weed control is not an effective way to increase economic returns (Gates, 2000). Most problematic is the lack of a selective herbicide to control grassy weeds in bahiagrass. The following weeds coestablished during our field trial: common bermudagrass, goosegrass, crabgrass, crowfootgrass, pigweed, teaweed, and wild radish. All these problematic weeds displayed 100% leaf necrosis following application of 0.3% glufosinate ammonium. This demonstrates that glufosinate ammonium resistant bahiagrass will work effectively in weed control programs and increase the value of this low-input turf and forage grass.

	ACKNOWLEDGMENTS

 
We would like to thank the USDA Biotechnology Risk Assessment Program for financial support, Dr. Maria Gallo (Agronomy Department, University of Florida) for critical reading of the manuscript, and Melissa Thorpe (NFREC, University of Florida) for photography displayed in Fig. 2g.

	NOTES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher.

Received for publication November 6, 2006.

	REFERENCES

TOP NOTES ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

• Agrawal, P.K., A. Kohli, R.M. Twyman, and P. Christou. 2005. Transformation of plants with multiple cassettes generates simple transgene integration patterns and high expression levels. Mol. Breed. 16:247–260.[CrossRef]
• Altpeter, F., and V.A. James. 2005. Genetic transformation of turf-type bahiagrass (Paspalum notatum Flügge) by biolostic gene transfer. Int. Turfgrass Soc. Res. J. 10:485–489.
• Altpeter, F., V. Vasil, V. Srivastava, E. Stoeger, and I.K. Vasil. 1996. Accelerated production of transgenic wheat (Triticum aestivum L.) plants. Plant Cell Rep. 16:12–17.[CrossRef][Web of Science]
• Bevan, M. 1984. Binary Agrobacterium vectors for plant transformation. Nucleic Acids Res. 12:8711–8721.[Abstract/Free Full Text]
• Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72:248–254.[CrossRef][Web of Science][Medline]
• Breitler, J.C., A. Labeyrie, D. Meynard, T. Legavre, and E. Guiderdoni. 2002. Efficient microprojectile bombardment- mediated transformation of rice using gene cassettes. Theor. Appl. Genet. 104:709–719.[CrossRef][Web of Science][Medline]
• Burton, G.W. 1948. The method of reproduction in common bahiagrass, Paspalum notatum. J. Am. Soc. Agron. 40:443–452.
• Castle, L.A., G.S. Wu, and D. McElroy. 2006. Agricultural input traits: Past, present, and future. Curr. Opin. Biotechnol. 17:105–112.[Web of Science][Medline]
• Chambliss, C.G., and M.B. Adjei. 2006. Bahiagrass. SS-AGR-36. Agron. Dep., Univ. of Florida, Gainesville.
• Christensen, A.H., and P.H. Quail. 1996. Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res. 5:213–218.[CrossRef][Web of Science][Medline]
• Dixon, L., T. Nyffenegger, G. Delley, J. Martinezizquierdo, and T. Hohn. 1986. Evidence for replicative recombination in cauliflower mosaic virus. Virology 150:463–468.[CrossRef][Web of Science]
• Fu, X., L.T. Duc, S. Fontana, B.B. Bong, P. Tinjuangjun, D. Sudhakar, R.M. Twyman, P. Christou, and A. Kohli. 2000. Linear transgene constructs lacking vector backbone sequences generate low-copy-number transgenic plants with simple integration patterns. Transgenic Res. 9:11–19.[CrossRef][Web of Science][Medline]
• Gao, C.X., L. Jiang, M. Folling, L.B. Han, and K.K. Nielsen. 2006. Generation of large numbers of transgenic Kentucky bluegrass (Poa pratensis L.) plants following biolistic gene transfer. Plant Cell Rep. 25:19–25.[CrossRef][Web of Science][Medline]
• Gates, R.N. 2000. Response of incomplete Tifton 9 bahiagrass stands to renovation. J. Range Manage. 53:614–616.
• Gates, R.N., C.L. Quarin, and C.G.S. Pedreira. 2004. Bahiagrass. p. 651–680. In L.E. Moser et al. (ed.) Warm-season (C₄) grasses. Agronomy Monogr. 45. ASA, CSSA, and SSSA, Madison, WI.
• Goldman, J.J., W.W. Hanna, G.H. Fleming, and P. Ozias-Akins. 2004. Ploidy variation among herbicide-resistant bermudagrass plants of cv. TifEagle transformed with the bar gene. Plant Cell Rep. 22:553–560.[CrossRef][Web of Science][Medline]
• Gondo, T., S. Tsuruta, R. Akashi, O. Kawamura, and F. Hoffmann. 2005. Green, herbicide-resistant plants by particle inflow gun-mediated gene transfer to diploid bahiagrass (Paspalum notatum). J. Plant Physiol. 162:1367–1375.[CrossRef][Web of Science][Medline]
• Hartman, C.L., L. Lee, P.R. Day, and N.E. Tumer. 1994. Herbicide-resistant turfgrass (Agrostis palustris Huds.) by biolistic transformation. Bio-Tech. 12:919–923.[CrossRef]
• Li, L., R. Li, S. Fei, and R. Qu. 2005. Agrobacterium-mediated transformation of common bermudagrass (Cynodon dactylon). Plant Cell Tissue Organ Cult. 83:223–229.[CrossRef][Web of Science]
• Luo, H., Q. Hu, K. Nelson, C. Longo, A.P. Kausch, J.M. Chandlee, J.K. Wipff, and C.R. Fricker. 2004. Agrobacterium tumefaciens-mediated creeping bentgrass (Agrostis stolonifera L.) transformation using phosphinothricin selection results in a high frequency of single-copy transgene integration. Plant Cell Rep. 22:645–652.[CrossRef][Web of Science][Medline]
• Meyer, P. 2000. Transcriptional transgene silencing and chromatin components. Plant Mol. Biol. 43:221–234.[CrossRef][Web of Science][Medline]
• Murashige, T., and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473–497.[CrossRef]
• Murray, M.G., and W.F. Thompson. 1980. Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res. 8:4321–4325.[Abstract/Free Full Text]
• Odell, J.T., F. Nagy, and N.H. Chua. 1985. Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810–812.[CrossRef][Medline]
• Rochester, D.E., J.A. Winer, and D.M. Shah. 1986. The structure and expression of maize genes encoding the major heat shock protein, hsp70. EMBO J. 5:451–458.[Web of Science][Medline]
• Sambrook, J., and D.W. Russell. 2001. Molecular cloning: A laboratory manual. Cold Spring Harbor Lab. Press, Cold Spring Harbor, NY.
• Sanford, J.C., E.D. Wolf, and N.K. Allen. 1990. Method for transporting substances into living cells and tissues and apparatus therefor. U.S Patent 4945050. Date issued: 31 July.
• Smith, R.L., M.F. Grando, Y.Y. Li, J.C. Seib, and R.G. Shatters. 2002. Transformation of bahiagrass (Paspalum notatum Flugge). Plant Cell Rep. 20:1017–1021.[CrossRef][Web of Science]
• Thompson, C.J., N.R. Movva, R. Tizard, R. Crameri, J.E. Davies, M. Lauwereys, and J. Botterman. 1987. Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus. EMBO J. 6:2519–2523.[Web of Science][Medline]
• Wang, M.B., and P.M. Waterhouse. 2000. High-efficiency silencing of a beta-glucuronidase gene in rice is correlated with repetitive transgene structure but is independent of DNA methylation. Plant Mol. Biol. 43:67–82.[CrossRef][Web of Science][Medline]
• Wild, A., and C. Wendler. 1993. Inhibitory action of glufosinate ammonium on photosynthesis. Z. Naturforsch. C: Biosci. 48:367–373.
• Zhang, G., S. Lu, T.A. Chen, C.R. Funk, and W.A. Meyer. 2003. Transformation of triploid bermudagrass (Cynodon dactylon x C. transvaalensis cv. TifEagle) by means of biolistic bombardment. Plant Cell Rep. 21:860–864.[Web of Science][Medline]

This article has been cited by other articles:

				G. Luciani, F. Altpeter, J. Yactayo-Chang, H. Zhang, M. Gallo, R. L. Meagher, and D. Wofford Expression of cry1Fa in Bahiagrass Enhances Resistance to Fall Armyworm Crop Sci., November 7, 2007; 47(6): 2430 - 2436. [Abstract] [Full Text] [PDF]

This Article Abstract Figures Only Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Sandhu, S. Articles by Blount, A. R. Search for Related Content PubMed Articles by Sandhu, S. Articles by Blount, A. R. Agricola Articles by Sandhu, S. Articles by Blount, A. R. Related Collections Forage Management Turfgrass Management Turfgrass Pesticides