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a USDA-ARS-PWA, Western Regional Research Center, 800 Buchanan St., Albany, CA 94710-1105
b Zabinskiego 18, Apt. 30, 02-793 Warsaw, Poland
c Dep. of Plant Sciences and Landscape Systems, Univ. of Tennessee, 2431 Center Drive, Knoxville, TN 37996-4561
* Corresponding author (congerbv{at}utk.edu)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2 test at P = 0.05. These results indicate that the Agrobacterium method is effective for transferring foreign genes into switchgrass.
Abbreviations: Act1, actin 1 promoter • bar, phosphinothricin acetyltransferase gene • GA3, gibberellic acid • gfp, green fluorescent protein gene • gus, ß-glucuronidase gene • kb, kilobase • MS, Murashige and Skoog medium • PCR, polymerase chain reaction • Ubi1, ubiquitin 1 promoter • X-Gluc, 5-bromo-4-chloro-3-indolyl-3-glucuronic acid
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The current status of forage and turf grass biotechnology has been recently reviewed (Forster and Spangenberg, 1999; Spangenberg et al., 2001; Wang et al., 2001). Transgenic plants have been reported for only 6 genera and 11 species (Wang et al., 2001); the only warm season grass listed is switchgrass. This was achieved in our laboratory by microprojectile bombardment of embryogenic calluses with a GFP-BAR plasmid (Richards et al., 2001). Integration of both the green fluorescent protein (gfp) and bar genes was shown by Southern blot hybridization. Fluorescing pollen was observed in T0 plants and the bar gene was transmitted through both male and female gametes and expressed in T1 progeny.
Microprojectile bombardment and gene transfer by A. tumefaciens are currently the two most common methods for achieving genetic transformation in higher plants. Although not universally accepted (Smith et al., 2001), it has been reported that Agrobacterium-mediated transformation leads to clean, discrete, low copy, well-defined, unrearranged DNA insertions into the plant genome (Chilton, 1993; Repellin et al., 2001; Upadhyaya et al., 2000). There are now several publications describing genetic transformation in various cereal species using Agrobacterium (Repellin et al., 2001). The only previous report of gene transfer in a forage or turf grass by this method was for GFP in creeping bentgrass {Agrostis palustris Huds. [= A. stolonifera var. palustris (Huds.) Farw.]; Yu et al., 2000}.
The objectives of the present study were to demonstrate high efficiency Agrobacterium-mediated transformation in switchgrass, and to show sexual transmission of the transgenes and their expression in T1 progeny. The accomplishment of such provides an alternative to microprojectile bombardment for genetic manipulation of this species.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Agrobacterium was grown from a single colony in MG/L medium (Garfinkel and Nester, 1980) supplemented with 20 mg L-1 rifampicin and 5 mg L-1 tetracycline at 27°C for 40 h. Aliquots of this culture (200 µL) were mixed with 200 µL of 15% aqueous glycerol and stored at -80°C. Bacteria were grown in 10 mL MG/L medium (with or without acetosyringone) at 27°C for 24 h and the resultant cultures (OD600 0.560) were used for transformation.
Inoculation and Cocultivation
Approximately 25 immature somatic embryos collected from embryogenic callus or 10 to 15 callus pieces (2 by 2 mm each) were transferred into 1.5 mL of A. tumefaciens suspension in multiwell plates and incubated at 27°C for various periods (3–60 min) in the dark. After inoculation, the explants were transferred with a wide-mouth pipette into Petri dishes containing 25 mL of the MS medium described above. Cocultivation was performed at 27°C in the dark for 3 to 5 d.
Basal parts (5–6 mm) of plantlets, obtained from somatic embryos, were cut transversely into smaller pieces (2–3 mm) in the presence of A. tumefaciens and inoculated in the bacterial suspension at 27°C in the dark for 1 h. In some experiments, explants were precultured on callus induction medium for 5 or 10 d. Both uncultured and precultured segments were wounded with carborundum before inoculation. Ten segments were placed in a test tube containing 10 mg carborundum in 5 mL of liquid MS medium without plant growth regulators and vortexed at a low speed for 1 min. Cocultivation was performed on the induction medium at 27°C in the dark for 3 to 7 d.
Mature caryopses were dehusked in 60% H2SO4 and sterilized with commercial bleach as previously described (Denchev and Conger, 1994). Caryopses were infected with A. tumefaciens by incubation in the bacterial suspension at 27°C for 1 h in the dark followed by transfer to callus induction medium and coculture for 3 to 5 d. Caryopses were also precultured on callus induction medium for 2 wk. Explants with formed callus were infected with Agrobacterium using the procedure for embryogenic callus.
In all experiments, the virulent system of the bacterium was stimulated by addition of 50 or 200 µM acetosyringone to the media for inoculation and/or cocultivation of somatic embryos and embryogenic callus. Mature caryopses and plantlet segments were infected with Agrobacterium cultures grown in the presence of 100 µM acetosyringone.
ß-Glucuronidase Activity Assay
ß-Glucuronidase activity was assayed histochemically in explants and callus tissues after cocultivation as well as after 1 wk culture on the selection medium by incubation for a period of 16 h in a 5-bromo-4-chloro-3-indolyl-3-glucuronic acid (X-Gluc) solution as described by Jefferson et al. (1987). Twenty percent methanol was added to eliminate endogenous GUS activity (Kosugi et al., 1990). Explants stained for GUS activity immediately after cocultivation were incubated in a liquid MS medium supplemented with 150 mg L-1 Timentin overnight to eliminate the Agrobacterium before incubation in the X-Gluc solution. ß-Glucuronidase expression was also examined in leaf tissue, tillers, and floral organs (anthers and ovaries).
Selection of Bialaphos-Resistant Callus Lines and Putative Transformants
Following cocultivation with Agrobacterium, the explants were transferred to callus induction medium containing 150 mg L-1 Timentin to eliminate the bacterium. Three different bialaphos selection schemes were evaluated. In the first method, tissues were cultured for 7 to 10 d without bialaphos before transfer to selection medium containing 10 mg L-1 bialaphos. In the second method, cocultured tissues were first placed on MS medium supplemented with 3 mg L-1 bialaphos and then transferred to MS medium containing 10 mg L-1 bialaphos. The third selection method involved transferring explants directly onto medium with 10 mg L-1 bialaphos immediately after cocultivation with the Agrobacterium. All explants were maintained on the medium containing 10 mg L-1 bialaphos for 4 wk with a biweekly subculture. During subculture, each piece of callus derived from one explant or one piece of inoculated callus was divided into several small pieces. Vigorously growing calluses were transferred to a regeneration medium (MS with 1.4 µM GA3) containing 10 mg L-1 bialaphos.
Resultant plantlets were tested for their response to Basta at different stages of growth by rubbing the leaves with sterile Q-tips soaked with a 0.1% solution of the herbicide. The first treatment was applied in vitro when the plantlets were growing on the selection medium and had 3 to 4 leaves. Two weeks after the treatment, the number of Basta-tolerant and Basta-sensitive plantlets obtained from each explant was determined and the percentage of the escapes was calculated. Plantlets showing no reaction to the herbicide were transferred to Magenta boxes (Sigma Chemical Co., St. Louis, MO) containing MS medium without the selection agent. They were again tested for their reaction to Basta at the transfer to 1-L polypropylene culture vessels (Phytacon, Sigma Chemical Co.) containing the same medium. After another 2 to 3 wk of culture, transformation frequency was determined as the number of tolerant plants recovered per explant inoculated. More than 100 plants were treated with Basta for the third time and transferred to moist peat pellets. Sixty plants were randomly selected, established in soil, and transferred to greenhouse benches. They received a final Basta treatment in the greenhouse by rubbing at least one leaf of each tiller with 0.1% of the herbicide.
Molecular Analyses
Genomic DNA of control, T0, and T1 plants was isolated from 250 mg of young leaf tissue using the Puregene DNA Isolation Kit (Gentra Systems, Minneapolis, MN). Polymerase chain reaction (PCR) was used to screen 30 putative T0 plants for presence of both the gus and bar genes. Primer sets used were: 5'-CAGGAAGTGATGGAGCATCAG-3' and 5'-TCGTGCACCATCAGCACGTTA-3' for gus (Becker et al., 1994), and 5'-CTGAAGTCCAGCTGCCAGAA-3' and 5'-CATCGTCAACCACTACATCG-3' for bar (Denchev et al., 1997). On the basis of this screening, 21 plants were selected for Southern blot hybridization. A PCR analysis to test for the presence of a conserved region of the virC gene of Agrobacterium was also performed on these 21 plants using the primers 5'-ATGATTTGTAGCGGACT-3' and 5'-AGCTCAACCTGCTTC-3' (Sawada et al., 1995). The predicted sizes of the amplified DNA fragments were 438 bp, 637 bp, and 730 bp for bar, gus, and virC, respectively. The PCR conditions were as previously described (Denchev et al., 1997; Sawada et al., 1995).
Southern blot analyses were performed with 15 µg of DNA digested with the restriction enzymes BamHI and SpeI in separate experiments. Digested DNAs were size-fractionated by agarose gel electrophoresis and transferred to a Hybond-N nylon membrane following the manufacturer's protocol (Amersham Pharmacia Biotech). The digoxigenin-labeled bar and gus probes for hybridization of BamHI-digested DNA from primary transformants were generated by PCR amplification (PCR DIG Probe Synthesis Kit, Roche Molecular Biochemicals, Indianapolis, IN) according to Tingay et al. (1997). Membrane hybridization and post-hybridization washes were conducted as described by Engler-Blum et al. (1993). Detection of the digoxigenin-hybridized fragments was performed by enzyme immunoassay and enzyme-catalyzed color reaction (DIG Nucleic Acid Detection Kit, Roche Molecular Biochemicals) as specified by the manufacturer. For hybridization of SpeI-digested DNA, a 1.8-kb KpnI-NcoI fragment representing the gus gene and a 0.54-kb KpnI-PstI fragment containing the bar gene isolated from the plasmid pDM805 were used as templates for the probe synthesis. The [32P]-labeled probes were obtained by using the RadPrime DNA Labeling System (Invitrogen Corp., Carlsbad, CA), according to the manufacturer's instructions.
PCR analyses for presence of the gus and bar genes in T1 progeny were performed as previously outlined for T0 plants. To confirm that the amplified fragments corresponded to the two genes, the PCR gels were blotted for Southern analysis and hybridized with [32P]-labeled probes as described above.
Progeny Analysis
Controlled crosses were made between transgenic plants, used as both male and female parents, and control Alamo plants. T1 offspring were assayed for Basta tolerance by rubbing the seedling leaves with a 0.1% solution of the herbicide and for GUS by using the histochemical assay of leaf tissues. Data were analyzed by the 2 test at P = 0.05.
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RESULTS |
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Effect of Acetosyringone on Transformation Efficiency
Acetosyringone did not affect the frequency of GUS expression when added to the media for inoculation and/or cocultivation. However, the presence of this compound increased the recovery of transgenic plants from most of the explants used. Data for the effect of acetosyringone on transformation efficiency of somatic embryos and established embryogenic calluses selected by the three methods described are summarized in Tables 2, 3, and 4
. The highest transformation efficiency in Alamo genotype C50 was obtained when somatic embryos were both inoculated and cocultivated in the presence of 200 µM acetosyringone (Table 2). Some of the somatic embryos transformed without acetosyringone also formed calluses during subsequent selection, but none produced transgenic plantlets. The highest number of Basta-tolerant plantlets produced from embryogenic calluses was obtained using media without acetosyringone either during inoculation or cocultivation. When 200 µM acetosyringone was added to the inoculation medium, overgrowth of the bacterium was observed during the selection, which resulted in inhibition of callus growth. Data for transformation efficiency of somatic embryos and established callus cultures from other Alamo genotypes are shown in Tables 3 and 4, respectively. A total of 2550 calluses and 830 somatic embryos were used in the experiments. Only genotypes that produced at least one transgenic plant are included in the data presented. Depending on genotype, explant used, and acetosyringone concentration, transformation efficiencies ranged from 0 to nearly 100%. In general, transformation efficiency was higher with somatic embryos than with calluses.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The highest efficiency for recovery of transgenic plants was obtained when embryogenic calluses and somatic embryos were used as targets for infection. Of these, somatic embryos were superior because they proliferated highly embryogenic callus, each of which produced numerous transformed plants during the selection process. Also, most of the recovered plantlets were transgenic, whereas 
30% of the plantlets obtained from infected calluses were untransformed escapes.
Acetosyringone increased the frequency of transgenic plants recovered, especially from somatic embryos, and improved the efficiency in most of the genotypes utilized. These results are in agreement with most reports of Agrobacterium-mediated transformation of Poaceae species. Our obtainment of transgenic plants without acetosyringone is in disagreement with Azhakanandam et al. (2000), who reported that it was not possible to induce transient GUS expression in rice embryogenic callus in the absence of this compound. Our transformation efficiencies are among the highest reported for the Agrobacterium method in grass and cereal species and are higher than the 10 to 31% reported by Akhakanandam et al. (2000) for rice. The success is probably due to the virulence of the Agrobacterium strain (AGL 1) used in our experiments and the susceptibility of the target tissues. Also, application of Basta to young plantlets allowed early elimination of most of those that were untransformed. Only a few escapes were identified after the second herbicide treatment.
Another significant factor contributing to the success is the promoters in pDM805 driving transgene expression. The maize Ubi1 promoter is used for the bar gene and the rice actin (Act1) promoter for the gus gene. These are the same promoters that are in the GFP-BAR plasmid used in our previous switchgrass transformation with microprojectile bombardment (Richards et al. 2001). Generally, in most monocot systems, Ubi1 has proved to be the strongest promoter followed by Act1 (Li et al., 1997; McElroy and Brettell, 1994).
Although the genotypes used were all within the cultivar Alamo, there were differences for recovering transgenic plants. Genotype differences for Agrobacterium-mediated transformation frequencies have also been reported for various Poaceae species, for example, different rice cultivars (Azhakanandam et al., 2000; Ke et al., 2001) and maize inbred lines (Lupotto et al., 1999).
Inoculation in the bacterial suspension for 3 to 60 min did not cause significant changes in the frequency of transgenic plants recovered. However, longer inoculation periods resulted in overgrowth of the Agrobacterium. Optimum cocultivation periods depended on target tissue used and were shorter for somatic embryos than for other explants. Transfer of infected explants to medium with low or no bialaphos to favor multiplication of transformed cells resulted in more vigorously growing calluses, but did not enhance transformation efficiency. Rather, it allowed for more untransformed escapes.
Southern blot hybridization after digesting the genomic DNA with BamHI showed different hybridization patterns among all of the T0 plants tested, indicating that T-DNA was integrated into the plant genome. The results obtained with SpeI, since this enzyme cuts the plasmid at only one site, provides further and even stronger evidence of transgene integration into the host genome. On the basis of the DNA gel blot hybridizations, 60% of the analyzed plants had single inserts, which is close to the 60 to 70% observed in maize (Ishida et al., 1996) and significantly greater than the 32% for rice (Hiei et al., 1994) and the 35% for wheat (Cheng et al., 1997). In the Agrobacterium-mediated transformation study with creeping bentgrass (Yu et al., 2000), two plants derived from the same callus showed two identical insertions and two other plants from a different callus showed only one insertion.
The high proportion of discrete integration events containing single or low T-DNA copy numbers is considered a desirable characteristic of Agrobacterium for gene transfer. However, multiple copies have been reported in transgenic plants of various plant species using this method (Smith et al., 2001). Only two T0 switchgrass plants showed multiple insertions (3–8). The difference in the number of the bands detected in DNA from T0 Plant 14 after digestion with BamHI and SpeI suggests a rearrangement of some of the inserts, which often occurs in primary transformants containing multiple insert copies. The very weak GUS activity observed in leaf tissues and pollen of this plant suggests the possibility of gene silencing. High copy numbers also resulted in weak GFP expression in pollen of plants in our previous switchgrass transformation study (Richards et al., 2001). The other plants tested, which contained only one to two gene copies, showed stronger GUS expression.
Although progeny analyses have been reported for various transformation experiments with major cereals, there are only two reports of such with forage or turf grass species, switchgrass (Richards et al., 2001), and ryegrass (Lolium spp.) (Wang et al., 1997). This is probably because most of these grasses are self-incompatible and controlled pollinations must be made under controlled conditions. Although not stated, it appeared that the crosses made with ryegrass utilized the transgenic plants only as female parents; therefore, transmission of the transgenes through the male gametes was not demonstrated. The segregation ratios in the offspring obtained from crosses in the present study, showed the expected 1:1 for both traits when the transgenic plants were used as either the male or female parent. These data, combined with the PCR analyses of progeny, provide further confirmation for stable genetic transformation in these experiments.
In conclusion, Agrobacterium-mediated transformation has been demonstrated for switchgrass. Furthermore, the transgenes were sexually transmitted through both male and female gametes and expressed in T1 progeny. Tolerance to the herbicide Basta represents transfer of a gene of practical agronomic importance. Basta is sold commercially under the name Liberty, and Liberty Link products are of high current interest because they represent an alternative to Roundup Ready as crops tolerant to a nonselective herbicide.
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ACKNOWLEDGMENTS |
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Received for publication January 28, 2001.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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