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Stable expression of AtGA2ox1 in a low-input turfgrass (Paspalum notatum Flugge) reduces bioactive gibberellin levels and improves turf quality under field conditions

Authors

  • Mrinalini Agharkar,

    1. Agronomy Department, Plant Molecular Biology Program, Genetics Institute, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611, USA
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  • Paula Lomba,

    1. Agronomy Department, Plant Molecular Biology Program, Genetics Institute, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611, USA
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  • Fredy Altpeter,

    Corresponding author
      * Correspondence (fax 352 392 7248; e-mail faltpeter@ifas.ufl.edu)
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  • Hangning Zhang,

    1. Agronomy Department, Plant Molecular Biology Program, Genetics Institute, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611, USA
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  • Kevin Kenworthy,

    1. Agronomy Department, Plant Molecular Biology Program, Genetics Institute, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL 32611, USA
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  • Theo Lange

    1. Institute for Plant Biology, Technical University of Brunswick, Mendelssohnstraße 4, D-38106 Braunschweig, Germany
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* Correspondence (fax 352 392 7248; e-mail faltpeter@ifas.ufl.edu)

Summary

Bahiagrass (Paspalum notatum Flugge) is a prime candidate for molecular improvement of turf quality. Its persistence and low input characteristics made it the dominant utility turfgrass along highways in the south-eastern USA. However, the comparatively poor turf quality due to reduced turf density and prolific production of unsightly inflorescences currently limits the widespread use of bahiagrass as residential turf. Alteration of endogenous gibberellin (GA) levels by application of growth regulators or transgenic strategies has modified plant architecture in several crops. GA catabolizing AtGA2ox1 was subcloned under the control of the constitutive maize ubiquitin promoter and Nos 3’UTR. A minimal AtGA2ox1 expression cassette lacking vector backbone sequences was stably introduced into apomictic bahiagrass by biolistic gene transfer as confirmed by Southern blot analysis. Expression of AtGA2ox1 in bahiagrass as indicated by reverse transcription–polymerase chain reaction and Northern blot analysis resulted in a significant reduction of endogenous bioactive GA1 levels compared to wild type. Interestingly, transgenic plants displayed an increased number of vegetative tillers which correlated with the level of AtGA2ox1 expression and enhanced turf density under field conditions. This indicates that GAs contribute to signalling the outgrowth of axillary buds in this perennial grass. Transgenic plants also showed decreased stem length and delayed flowering under controlled environment and field conditions. Consequently, turf quality following weekly mowing was improved in transgenic bahiagrass. Transgene expression and phenotype were transmitted to seed progeny. Argentine bahiagrass produces seeds asexually by apomixis, which reduces the risk of unintended transgene dispersal by pollen and results in uniform progeny.

Introduction

Bahiagrass is an important forage and turf species in the south-eastern USA and other subtropical regions. The popularity of bahiagrass can be attributed to its tolerance to heat, low fertility, drought, mowing and overgrazing. It is also resistant to most diseases and pests. In spite of these benefits, bahiagrass is primarily used as forage and utility turf along highways and for low-quality residential lawns. Compared to other turfgrasses, bahiagrass displays a less dense turf that is a consequence of reduced tillering (Anowarul Islam and Hirata, 2005), which severely limits its turf quality. The reduction of length and number of inflorescences are also desirable to reduce the need for frequent mowing (Goatley et al., 1998).

The habit of bahiagrass is established by the initiation and outgrowth of buds forming new tillers. Two developmental processes are negatively correlated with the production of vegetative tillers: apical dominance and the formation of inflorescence-bearing, reproductive tillers (Jewiss, 1972). Argentine bahiagrass produces tall, unsightly inflorescences with increased temperatures and long days.

Gibberellins (GA) are plant hormones that are essential endogenous regulators of plant growth and development (Harberd et al., 1998; Pimenta Lange and Lange, 2006). The manipulation of bioactive GA levels in plants thus effectively alters plant architecture (Hedden and Phillips, 2000; Pimenta Lange and Lange, 2006; Wang and Li, 2006). Only a few of these GAs are biologically active, including GA1, GA3, GA4, GA5, GA6, and GA7 (Sponsel and Hedden, 2004), with GA1 being the most abundant biologically active GA in bahiagrass (T. Lange, unpublished). Bioactive GAs have been proposed to suppress tiller bud outgrowth, enhance apical dominance and promote the formation of inflorescence development under long days in grasses (Lester et al., 1972; Johnston and Jeffcoat, 1977; MacMillan et al., 2005). Trinexapac-ethyl (Primo) is a plant growth regulator that inhibits GA synthesis. Multiple applications of trinexapac-ethyl seem to favour quicker turf establishment supported by more intensive tillering (Ervin and Koski, 1998). Treatment of turf with trinexapac-ethyl consistently for three growing seasons substantially decreased mowing requirement, while displaying better visual quality than untreated turf (Lickfeldt et al., 2001). To reduce rising mowing costs, plant growth regulators are used routinely on bahiagrass growing on roadsides, residential areas and golf course rough areas (Unruh and Brecke, 1999). However, the need for frequent applications and the potential for phytotoxicity pose problems (Unruh and Brecke, 1999). In view of this, and in the absence of a dwarf bahiagrass cultivar, the use of a molecular approach to improve the turf quality of bahiagrass is desirable.

The first step of GA degradation involves GA 2-oxidases that hydroxylate carbon-2 of GAs (Martin et al., 1999; Thomas et al., 1999; Sakamoto et al., 2001). GA 2-oxidases from several species have been cloned, including runner bean (Thomas et al., 1999), garden pea (Lester et al., 1999; Martin et al., 1999), Arabidopsis (Thomas et al., 1999; Hedden and Phillips, 2000; Schomburg et al., 2003), rice (Sakamoto et al., 2001; Sakamoto et al., 2003), poplar (Busov et al., 2003), and pumpkin (Frisse et al., 2003). GA 2-oxidases are multifunctional 2-oxoglutarate-dependent dioxygenases and typically catalyse the 2β-hydroxylation of C19-GAs. GA 2-oxidases including At-GA2ox1 have been shown to convert GA9 and GA20, the immediate precursors of bioactive GA4 and GA1 into biologically inactive GA51 and GA29, respectively (Thomas et al., 1999). They also hydroxylate carbon-2 of bioactive GAs, GA1 and GA4 to GA8 and GA34, respectively (Martin et al., 1999; Thomas et al., 1999) and render them inactive. In addition, GA 2-oxidases, which do not use C19-GAs as their substrates but are able to catalyse the 2β-hydroxylation of the C20-GA precursors GA12 and GA53, have also been described (Schomburg et al., 2003). Some GA-oxidase gene expression is controlled by feedback or feed-forward types of regulation mechanisms that are activated in the plant when bioactive GA levels are altered (Pimenta Lange and Lange, 2006). The use of a strong promoter was effective to overrule such possible feedback mechanisms (Radi et al., 2006).

An efficient protocol for tissue culture and transformation of the apomictic cultivar Argentine of bahiagrass has been recently developed in our laboratory (Altpeter and James, 2005; Altpeter and Positano, 2005) and now allows the introduction of heterologous GA catabolizing enzymes. The heterologous expression of GA 2-oxidases resulted in the production of plants with reduced height and delayed flowering in wheat (Hedden and Phillips, 2000) and rice (Sakamoto et al., 2001). We describe here the stable expression of AtGA2-ox1 in bahiagrass and its correlation to bioactive GA1 levels and turf quality under controlled environment and field conditions.

Results

Transformation and molecular characterization

Biolistic gene transfer of minimal nptII and AtGA2ox1 expression cassettes (Figure 1a) into 600 calli resulted in the regeneration of eight independent, paromomycin-resistant bahiagrass plants. NPTII ELISA confirmed the expression of nptII in all regenerated transgenic lines (data not shown), indicating that no non-transgenic plants escaped the selection process. Polymerase chain reaction (PCR) with AtGA2ox1 specific primers allowed the amplification of the expected product from all transgenic plants but not from the wild type (Figure 1b) indicating a 100% cotransformation frequency. Reverse transcription-PCR (RT-PCR) analysis (Figure 1c) confirmed the presence of the AtGA2ox1 transcripts in all lines. Southern blot analysis was carried out following restriction digestion of genomic DNA with BamHI which cuts once at the 5′ end of the AtGA2ox1 coding region. Ethidium bromide staining (not shown) confirmed equal loading of all DNA samples. Hybridization with a probe from the AtGA2ox1 coding region confirmed the independent nature of the transgenic lines (Figure 1d). The transgenic lines displayed a simple transgene integration pattern with two (H1, H2, H6) to four (H3) hybridization signals.

Figure 1.

Transformation and molecular characterization of transgenic bahiagrass plants. (a) Transgene expression cassette containing the maize ubiquitin promoter, AtGA2ox1 and Nos terminator. (b) PCR analysis using AtGA2ox1 specific primers for amplification of genomic DNA isolated from transgenic lines (L1, H1, H2, H3, H4, H5, H6 and H7) compared to wild type (WT), plasmid pHZUbiox1 (PC) and buffer (NC). (M) 2-Log DNA ladder (0.1–10 kb) (New England Biolabs). (c) RT-PCR using AtGA2ox1 specific primers for amplification of cDNA from transgenic lines (H1, H2, H3, H4, H5, H6, H7 and L1) compared to WT and plasmid pHZUbiox1 (PC) and buffer (NC). (M) 2-Log DNA Ladder (0.1–10 kb) (New England Biolabs). (d) Southern blot analysis of genomic DNA from the wild type (WT) and transgenic lines (H1, H2, H3, H6 and H7) following restriction digestion with BamHI. The linearized plasmid pHZUbiox1 (PC, 20 pg) was used as the positive control and the entire 1.2 kb coding sequence of the AtGA2ox1 was used as a probe. (e) Northern blot analysis of total RNA from transgenic lines (L1, H1, H2, H3, H4, H5, H6 and H7) and the WT hybridized with a probe of the AtGA2ox1 cDNA.

Northern blot analysis of transgenic lines revealed a clearly detectable AtGA2ox1 hybridization signal in all bahiagrass lines except L1 and wild type (Figure 1e). Methylene blue staining of the membrane confirmed equal loading of all samples (data not shown). Lines H1 and H6 showed the highest level of expression compared to lines H2, H3 and H4, which displayed a clearly detectable hybridization signal of lower intensity.

Quantification of endogenous GAs

The levels of the primary, bioactive GA in bahiagrass, GA1, its precursor (GA20) and the products of their catabolism (GA8 and GA29, respectively) were analysed in AtGA2ox1 expressing line H1 and wild type by gas chromatography-mass spectrometry (GC-MS) selected ion monitoring. Line H1 displayed 75% less GA1 and 30% less GA20 than wild type (P < 0.05; Table 1). Compared to wild type, line H1 contained 7.3 times more GA29, the 2β-hydroxylated form of GA20 (P < 0.05; Table 1). The content of GA8 (2β-hydroxylated GA1) did not differ significantly between wild type and transgenic line H1 (P < 0.05; Table 1).

Table 1.  Analysis of gibberellin content [ng/g DW (dry weight)] from line H1 and wild type
GAH1Wild type
  • *

    Different letters following means in the same row indicate significant difference at P < 0.05.

GA202.23 ± 0.36b*3.20 ± 0.39a
GA10.70 ± 0.14b2.80 ± 0.34a
GA80.13 ± 0.48a0.23 ± 0.05a
GA290.73 ± 0.05a0.10 ± 0.04b

Evaluation of growth characteristics of transgenic lines

Single, rooted tillers of uniform size were obtained from greenhouse grown plants and propagated in a constantly aerated hydroponics solution. The plants were evaluated for their growth characteristics 4 weeks after establishment in hydroponics (Figure 2a). Transgenic lines H1, H2, H3, H4 (H-lines) with clearly detectable AtGA2ox1 Northern hybridization signals (Figure 1e) were compared with wild type and transgenic line L1 with no detectable AtGA2ox1 Northern hybridization signal but detectable AtGA2ox1 transcript by the more sensitive RT-PCR (Figure 1c).

Figure 2.

Phenotypic comparison of AtGA2ox1 expressing bahiagrass and wild type in hydroponics or soil under controlled environment conditions. (a) AtGA2ox1 expressing bahiagrass (H1, H2, H3 and H4), compared to the wild type (WT) following 4 weeks of growth of single rooted tillers in hydroponics. (b) Number of tillers produced by AtGA2ox1 expressing bahiagrass (H1, H2, H3 and H4), transgenic bahiagrass with barely detectable AtGA2ox1 expression (L1) and WT following 4 weeks of growth of single rooted tillers in hydroponics. (c) Length of the tiller measured from the crown to the tip of the leaf of AtGA2ox1 expressing bahiagrass (H1, H2, H3 and H4), transgenic bahiagrass with barely detectable AtGA2ox1 expression (L1) and WT following 4 weeks of growth of single rooted tillers in hydroponics. (d) Root biomass of AtGA2ox1 expressing bahiagrass (H1, H2, H3 and H4), transgenic bahiagrass with barely detectable AtGA2ox1 expression (L1) and WT following 4 weeks of growth of single rooted tillers in hydroponics. (e) Length of the longest root of AtGA2ox1 expressing bahiagrass (H1, H2, H3 and H4), transgenic bahiagrass with barely detectable AtGA2ox1 expression (L1) and WT following 4 weeks of growth of single rooted tillers in hydroponics. (f) Comparison of AtGA2ox1 expressing line H1 and WT following 6 weeks of growth of single rooted tillers in soil. (g) Comparison of AtGA2ox1 expressing line H2 and WT following 6 weeks of growth of single rooted tillers in soil. (h) Comparison of AtGA2ox1 expressing lines H3 and WT following 6 weeks of growth of single rooted tillers in soil. (i) Comparison of AtGA2ox1 expressing line H4 and WT following 6 weeks of growth of single rooted tillers in soil. (j) Number of tillers produced by AtGA2ox1 expressing bahiagrass (H1, H2, H3 and H4) and the WT following 6 weeks of growth of single rooted tillers in soil. (k) Length of the tiller measured from the crown to the tip of the leaf of AtGA2ox1 expressing bahiagrass (H1, H2, H3 and H4) and the WT following 6 weeks of growth of single rooted tillers in soil. (l) Length of the longest leaf from the base to the tip of AtGA2ox1 expressing bahiagrass (H1, H2, H3 and H4) and the WT following 6 weeks of growth of single rooted tillers in soil. (m) Length of the stem from the crown to the base of the first leaf of AtGA2ox1 expressing bahiagrass (H1, H2, H3 and H4) and the WT following 6 weeks of growth of single rooted tillers in soil.

AtGA2ox1 expressing lines produced between 1.8 (H2) and 2.4 times (H1) the number of tillers of wild type (P < 0.05), while wild type did not differ significantly from line L1 (Figure 2b). AtGA2ox1 expressing lines displayed also 21% (H2; H4) to 34% (H1) shorter tillers than wild type (P < 0.05; Figure 2c) as well as significantly shorter leaves (data not shown). Up to 32% shorter stems (data not shown) contributed to the shorter tillers of the transgenic lines. Wild type and line L1 did not differ significantly in any of these parameters. The length of the longest root did not differ significantly among lines H1, H3, H4, L1 and wild type. The total biomass (data not shown) as well as root biomass (Figure 2d) or shoot biomass (data not shown) produced by the AtGA2ox1 expresing lines did not differ significantly from the biomass of wild type or L1. However, line H2 displayed a 37% shorter root length than wild type (Figure 2e).

AtGA2ox1 expressing lines grown in soil (Figure 2f–i) produced between 1.2 (H2; H3) and 2.0 times (H1) the number of tillers of wild type (P < 0.05; Figure 2j). AtGA2ox1 expressing lines displayed also 18% to 33% shorter tillers than wild type (P < 0.05; Figure 2k). Both, 14% to 34% shorter leaves (Figure 2l) and 27% to 45% shorter stems (P < 0.05; Figure 2m) contributed to the shorter tillers of the AtGA2ox1 expressing lines. Flowering in AtGA2ox1 expressing lines was delayed for approximately 2 weeks compared to the wild type.

Progeny analysis

Transgenic and wild type plants produced seeds under greenhouse conditions. The seed coats were physically removed to release dormancy of the freshly harvested seeds. Seedlings from transgenic lines H3, H4, H6 and wild type were grown for 4 weeks. Seedlings from lines H3 and H4 showed a 20% to 30% growth reduction and more tillers compared to wild type (data not shown). Seedlings from the highly AtGA2ox1 expressing line H6 displayed uniformly a more than 50% decreased height compared to wild type along with more tillers (Figure 3a). RT-PCR analysis revealed expression AtGA2ox1 in all analysed transgenic seedlings in contrast to wild type (Figure 3b).

Figure 3.

Evaluation of seed progeny and field performance of AtGA2ox1 expressing bahiagrass in comparison to wild type. (a) Comparison of the seed progeny of AtGA2ox1 expressing line H6 and wild type (WT) following 6 weeks of growth after germination. (b) RT-PCR using AtGA2ox1 specific primers for amplification of cDNA from progeny of transgenic lines H3 (3-1, 3-2), H4 (4-1, 4-2), H6 (6-1, 6-2) compared to the WT, plasmid pHZUbiox1 (PC) and buffer (NC). (M) 2-Log DNA ladder (0.1–10 kb) (New England Biolabs). (c) Number of tillers produced by AtGA2ox1 expressing lines (H1 and H3) and WT in a 10 × 10-cm area following 12 weeks of growth after establishment of field plots. (d) Comparison of AtGA2ox1 expressing line H1 and WT 4 weeks after establishment of field plots. (e) Dry weight of clippings produced by AtGA2ox1 expressing lines (H1 and H3) and WT following 4 weeks of growth after establishment of field plots. (f) Dry weight of clippings produced by AtGA2ox1 expressing lines (H1 and H3) and WT under a weekly mowing regime following 14 weeks after establishment of field plots. (g) National Turfgrass Evaluation Program ratings of turf quality of AtGA2ox1 expressing lines (H1 and H3) in comparison to WT immediately after mowing and 14 weeks after establishment of field plots. (h) A freshly mowed AtGA2ox1 expressing line H1 following weekly mowing at 8 cm cutting heights and 14 week after establishment of field plots. (i) A freshly mowed WT following weekly mowing and 14 weeks after establishment of field plots.

Field evaluation of turf quality

AtGA2ox1 expressing lines H1 and H3 and wild type plants were established at the UF-IFAS Plant Research and Education Center in Citra, Florida, under USDA-APHIS permit 05-364-01r in a randomized block design with a total of 24 replications, using four 8 × 8 × 7-cm grass plugs for initiation of each 1 × 1-m plot. The density of transgenic plants was evaluated following 12 weeks of growth in the field by counting the number of tillers in a randomly selected 10 × 10-cm area per plot. AtGA2ox1 expressing lines H1 and H3 had 1.8 times and 1.3 times the number of tillers than wild type (P < 0.05; Figure 3c), respectively. The higher number of tillers in the transgenic plants resulted in a higher turf density which caused a more erect growth pattern (Figure 3d; H1) compared to wild type with more prostrate growth and open growth habit (Figure 3d; WT). The erect growth pattern and the higher number of tillers in the transgenic lines contributed to 2.1 times (H1) and 1.5 times (H3) greater clipping weights in transgenic lines compared to the wild type plants, following 4 weeks of growth after transplanting (P < 0.05; Figure 3e). As compared to wild type, a significant difference in clipping weights was also observed in H1 following a weekly mowing schedule (P < 0.05; Figure 3f) while line H3 showed no significant difference in clipping weight (Figure 3f). Mowing quality was rated according to the National Turfgrass Evaluation Program guide. This evaluation indicated that the transgenic lines displayed a significantly higher turf quality after mowing compared to the wild type (Figure 3g). Line H1 had the highest rating for mowing quality (Figure 3g). This is reflected in the comparison of freshly mowed H1 to wild type (Figure 3h,i). Line H1 (Figure 3h) displayed a high turf density with no gaps in contrast to wild type (Figure 3i).

Emergence of inflorescences in transgenic lines H1 and H3 was delayed by 2–3 weeks compared to the wild type (Table 2). The number of inflorescences produced by both transgenic lines was significantly lower than from wild type (P < 0.05; Table 1). The length of inflorescence stems of lines H1 and H3 was on average 8 cm and 5 cm shorter than wild type, respectively (P < 0.05; Table 2).

Table 2.  Development of inflorescences in AtGA2ox1 expressing lines (H1, H3) and wild type bahiagrass under field conditions.
Record/lineH1H3Wild type
  • *

    Different letters following means in the same row indicate significant difference at P < 0.05.

8 August 2006
 Average number of inflorescences per plot0.00 ± 0.00b*0.04 ± 0.04b1.46 ± 0.33a
29 August 2006
 Average number of inflorescences per plot54.38 ± 5.52b48.75 ± 3.46b62.88 ± 3.82a
 Average length of stems of fully expanded inflorescences (cm)39.79 ± 0.97b42.13 ± 1.46b47.10 ± 1.36a

Discussion

We report here for the first time a transgenic approach that resulted in improved turf quality of a low-input grass (Paspalum notatum Flugge) under field conditions. We stably introduced a constitutive expression cassette of AtGA2ox1, a GA 2-oxidase from Arabidopsis, into bahiagrass. Biolistic co-transfer of the minimal AtGA2ox1 and selectable marker cassettes, following removal of the vector backbone, resulted in 100% co-integration and co-expression frequency of the transgenes along with a simple transgene integration pattern. Minimal vector technology has been described earlier to reduce complexity of transgene integration and enhance co-expression frequencies of co-transferred transgenes (Fu et al., 2000; Agrawal et al., 2005; reviewed by Altpeter et al., 2005). Transgenic bahiagrass constitutively overexpressing AtGA2ox1, a GA 2-oxidase from Arabidopsis, displayed 75% less GA1 and 30% less GA20 than wild type, while the 2β-hydroxylated metabolite GA29 was elevated 7.3-fold. This response is consistent with the earlier described substrate specificities of AtGA2ox1 (Thomas et al., 1999). Constitutive overexpression of OsGA2ox1 in rice (Sakamoto et al., 2001) caused very similar changes in GA levels with 75% less GA1 than wild type while the 2β-hydroxylated metabolites of GA20 and GA1 were elevated 6.8- and 2.5-fold, respectively.

Overexpression of AtGA2ox1 in bahiagrass resulted in semi-dwarf plants with 27% to 45% shorter stems, 18% to 34% shorter leaves, delayed flowering, and 8 cm shorter inflorescence stems compared to wild type plants. During field evaluation, the transgenic lines continued to maintain their semi-dwarf phenotype and produced significantly higher number of tillers which resulted in increased turf density. Reduced steminess and increased number of tillers per area significantly enhanced the quality of the transgenic bahiagrass under weekly mowing. Similar to our results, constitutive overexpression of AtGA2ox1 in tobacco resulted in significant shorter plants than wild type, while allowing the production of viable seeds (Biemelt et al., 2004). In contrast, rice constitutively overexpressing OsGA2ox1 suppressed formation of inflorescences completely (Sakamoto et al., 2001).

The role of GAs as a leaf-sourced stimulating hormonal signal for flowering under long-day photoperiod has been proposed in perennial ryegrass (MacMillan et al., 2005). Consistently, overexpression of AtGA2ox1 results in a more than 2-week delay in flowering in field-grown bahiagrass under long-day photoperiod. However, viable seeds were obtained from most of the transgenic lines, and transgene expression and phenotype were transmitted to seed derived progeny. Many of the warm season turfgrasses including bahiagrass are vegetatively propagated and lawns are established from sod. Nevertheless, availability of seeds, as observed in AtGA2ox1 overexpressing plants, can be desirable for establishment of turf in low-input situations. Argentine bahiagrass is an obligate apomict (Burton, 1948), which reduces the risk of unintended transgene dispersal by pollen and results in uniform progeny.

The production of tillers by the transgenic plants correlated with AtGA2ox1 expression and was significantly higher in several lines than the wild type under hydroponics, greenhouse and field conditions. The highest AtGA2ox1 expressing line produced approximately twice as many tillers as the wild type under controlled environment and field conditions. Interestingly, earlier reports on GA 2-oxidase overexpression in annual crops did not describe an enhanced number of vegetative tillers, consistent with the concept that GAs primarily control plant height (Hedden and Phillips, 2000; Sakamoto et al., 2001; Busov et al., 2003; Sakamoto et al., 2003; Schomberg et al., 2003; Biemelt et al., 2004; Radi et al., 2006). However, bioactive GAs have been proposed to suppress tiller bud outgrowth and enhance apical dominance in grasses (Lester et al., 1972; Johnston and Jeffcoat, 1977). Enhanced axillary bud outgrowth is usually attributed to loss of apical dominance. Increasing evidence suggests that auxin and cytokinins are not the exclusive effectors of apical dominance (Wang and Li, 2006). More intense tillering was also achieved by multiple applications of the plant growth regulator trinexapac-ethyl (Primo), which specifically inhibits the biosynthesis of bioactive GAs (Ervin and Koski, 1998). Overexpression of ATH1 in perennial ryegrass resulted in 56% to 69% decreased levels of GA1 and was accompanied by the outgrowth of normally quiescent lateral meristems into extra leaves and delayed development of inflorescences (van der Valk et al., 2004). Phytochrome B mutants in sorghum display elevated GA levels along with enhanced apical dominance (Foster et al., 1994), enhanced expression of the SbTB1 repressor of bud outgrowth (Kebrom et al., 2006) and absence of tillering. The mechanism by which phyB regulates SbTB1 abundance remains to be discovered (Kebrom et al., 2006). Our data suggest that the GA-signalling pathway, among other components (Wang and Li, 2005), is involved in outgrowth of axillary buds in perennial grasses.

In summary, overexpression of a heterologous GA 2-oxidase in bahiagrass resulted in reduction of endogenous bioactive GA levels and produced semi-dwarf plants with increased tillering, delayed flowering, and shorter inflorescences. Our data suggest that GAs control flowering and outgrowth of axillary buds in this perennial grass. Field evaluation of AtGA2ox1 expressing bahiagrass under weekly mowing demonstrated rapid establishment and significantly improved turf quality of this low input grass due to increased turf density.

Experimental procedures

Vector construction

The coding region of AtGA2ox1 (accession no. AJ132435) gene was amplified from Arabidopsis thaliana genomic DNA using the primers described by (Biemelt et al., 2004). The amplified 1.2 kb fragment was cloned in the pDrive vector (Qiagen, Valencia, CA, USA), sequenced and inserted into an expression vector between polyubiquitin (ubi) promoter from Zea mays (S94464; Christensen et al., 1992; Christensen and Quail, 1996) with polyubiquitin (ubi) mature mRNA and first intron enhancer sequence from Z. mays (S94464; Christensen et al., 1992) and Nos (nopaline synthase) 3’UTR (Bevan, 1983; Fraley et al., 1983) following excision with the restriction enzymes BamHI (New England Biolabs, Ipswich, MA, USA) and SacI (New England Biolabs) to create the vector pHZUbiox1 (Figure 1d).

Tissue culture and genetic transformation

Mature seeds of Argentine bahiagrass were used to induce callus for genetic transformation. The tissue culture and gene transformation procedures were as described by Altpeter and James (2005). For selective purpose, the gene nptII (neomycin phosphotransferase II) under transcriptional control of the enhanced 35S promoter from cauliflower mosaic virus (CaMV) (AF234315; Kay et al., 1985; Odell et al., 1985) with heat shock protein 70 (HSP70) intron from Z. mays (X03714, X03697; Rochester et al., 1986) and the CAMV35S polyadenylation signal (Dixon et al., 1986) was co-transformed with the AtGA2ox1 expression cassette. Following the strategy described by Fu et al. (2000) minimal transgene expression constructs containing only the expression cassettes without vector backbone were used for biolistic gene transfer. After restriction digest with NotI (New England Biolabs) and NotI/AlwNI (New England Biolabs), respectively, minimal pHZUbiox1 and selectable 35S-nptII-35S 3′UTR expression cassettes were isolated by gel electrophoresis, the corresponding band was excised and purified using the QIAquick® Gel Extraction Kit (Qiagen) to remove vector backbone sequences.

The AtGA2ox1 and nptII expression cassettes were mixed in a 2 : 1 ratio, and precipitated on a 1 : 1 mixture of 0.75 µm and 1.0 µm diameter gold particles and delivered to embryogenic calli of Argentine bahiagrass using a DuPontPDS-1000/He device (Sanford et al., 1991) at 1100 psi. Putative transgenic callus and plantlets were selected and regenerated on culture medium containing 50 mg/L paromomycin sulphate (Phytotechnology Laboratories, Shawnee Mission, KS, USA). Rooted transgenic plants were transferred to soil and propagated under controlled environment conditions at 27 °C/20 °C day/night with 12-h photoperiod and 500 mE m−2s−1 light.

NPTII ELISA assay

The transgenic nature of the regenerated plants was confirmed by NPTII ELISA assay (Agdia, Elkhart, IN, USA). Total protein was extracted from 100 mg of leaf tissue using the extraction buffer supplied with the kit. Concentration of total protein was estimated using the Bradford protein assay (Bradford, 1976). The ELISA assay was performed according to the manufacturer's instructions. Twenty micrograms of total protein was used from putative transgenic plant or wild type in comparison to a serial dilution of the NPTII standard supplied with the kit. Qualitative evaluation of NPTII expression was possible by visual comparison of colour development.

PCR and RT-PCR

Genomic DNA was extracted as described earlier Dellaporta et al. (1983). Seventy-five to 80 ng genomic DNA was used as a template for PCR. PCR was carried out in an Eppendorf Mastercycler (Eppendorf, Westbury, NY, USA) using the HotStarTaq® DNA Polymerase (Qiagen). Samples were denatured at 95 °C for 15 min; followed by 30 cycles at 95 °C for 30 s, 52.3 °C for 30 s, 72 °C for 1 min; and final extension at 72 °C for 10 min. PCR products were analysed by electrophoresis on a 1.2% agarose gel. The primer pair with sense 5′-GAACACGAGACCGTCGATTT-3′ and antisense 5′-CGGGCTTTTGAGAAGACTTG-3′ was designed to amplify a 189-bp fragment from the gene AtGA2ox1, the same primer pair was used for RT-PCR expression analysis.

For RT-PCR, total RNA was extracted from 100 mg leaves using the RNeasy® Plant Mini Kit (Qiagen) followed by treatment with RNAse-free DNase (Qiagen) to eliminate genomic DNA contamination. For cDNA synthesis the iScriptTM cDNA Synthesis Kit (Bio-Rad Laboratories, Hercules, CA, USA) was used with 1 µg of total RNA in a reaction volume of 20 µL. To detect the transcripts of the gene AtGA2ox1 by PCR, 2 µL of the cDNA was used as a template with the same primer pair as described above for PCR from genomic DNA.

Southern blot analysis

Total genomic DNA was isolated from leaves of transgenic and wild type plants as described by Saghai-Maroof et al. (1984). Restriction digestion with BamHI (New England Biolabs) of 20 µg genomic DNA, was followed by electrophoresis on a 1% agarose gel and blotting on to a Hybond-N + membrane (Amersham Biosciences, Piscataway, NJ, USA). As positive control 25 pg of pHZUbiox1 plasmid DNA were used, following linearization by restriction digestion with BamHI. The entire 1.2 kb coding sequence of AtGA2ox1 was excised with a BamHI/SacI digest and used as a probe. The probe was labelled with [a-32P] dCTP by random priming using the Prime-a-Gene® Labeling System (Promega, Madison, WI, USA). Hybridization and detection were performed according to the manufacturer's instructions.

Northern blot analysis

TRI reagent (Sigma-Aldrich, St Louis, MO, USA) was used for the isolation of total RNA from leaves of transgenic and wild type plants. The extraction was performed according to the manufacturer's instructions using 300 mg of leaf tissue. Ten micrograms total RNA was resolved on a formaldehyde agarose gel (1.2% agarose) and transferred to Hybond-N + membrane (Amersham). The primer pair sense 5′-AGAACACGAGACCGTCGATT-3′ and antisense 5′-GGAGGGACAGAGATCCATGA-3′ was used to amplify a 500-bp region from Arabidopsis cDNA for use as the probe following probe following labelling with [α-32P] dCTP by random priming using the Prime-a-Gene® Labeling System (Promega). Hybridization and detection were performed according to the manufacturer's instructions.

Quantification of endogenous gibberellins

For quantitative determination of endogenous GAs, emerging tillers approximately 3 cm in length were sampled. Frozen plant material (0.1 g dry weight) was pulverized under liquid nitrogen and spiked with 17,17-d2-GA standards (2 ng each; from Prof. L. Mander, Australian National University, Canberra, Australia). Samples were extracted, purified, derivatized, and analysed by combined GC-MS using selected ion monitoring as described elsewhere (Lange et al., 2005). Means represent four biological replications.

Evaluation of plant phenotypes under hydroponics growth conditions

Single tillers with newly formed, 2–3 cm long roots were transferred to hydroponics nutrient solution made weekly of 1.2% Boost and 0.3% Grow (Technaflora Plant Products, Port Coquitlam, BC, Canada), pH 6.0. Roots were fully submerged into aerated nutrient solution with approximately 80% oxygen saturation. Four weeks after the culture initiation, the number of tillers, length of roots, vegetative biomass and root biomass were measured. The experiment was conducted as randomized block with four replications in a walk-in growth chamber with 400 mEm−2S−1 light intensity, 16/8 h light/dark, and 28 °C/20 °C day/night temperature. Transgenic lines expressing the AtGA2ox1 according to Northern blot analysis (H1, H2, H3, H4) were compared with wild type and one transgenic line showing a PCR and RT-PCR amplification product with AtGA2ox1 specific primers but no detectable AtGA2ox1 hybridization signal following Northern blot analysis (L1).

Greenhouse evaluation of plant architecture

Single rooted tillers from transgenic plants expressing the AtGA2ox1 according to Northern blot analysis (H1, H2, H3, and H4) or wild type plants were transplanted per 8 × 8 × 7 cm pot with steam sterilized topsoil, randomized in 48 replications and propagated in the greenhouse. After a 6-week growth period, the following parameters were evaluated: number of tillers, tiller length (from crown to tip of the longest leaf), length of stem (from crown to first leaf) and leaf length (average length of the three longest leaves). The temperature was controlled with air conditioning to approximately 30 °C during day and 25 °C during night. Irrigation and fertilization was provided using an ebb and flow system with watering twice a day for 5 min. The day length was maintained at 14 h using 1000 Watt sodium vapor lights.

Progeny analysis

Inflorescences of transgenic lines were bagged following the dehiscence of anthers to prevent seed loss by shattering. Seeds were harvested 4 weeks after bagging. For germination, seeds of transgenic lines and wild type were soaked overnight in distilled water and the glumes were removed with a scalpel to break the dormancy. The seeds were then soaked overnight in distilled water and transferred the following day to Fafard no. 2 mix (Conrad Fafard, Agawam, MA, USA). Tiller length and number of tillers were recorded six week after germination. Leaf tissue was harvested 6 weeks after germination. RNA isolation and RT-PCR was carried out as described above.

Field evaluation of turf quality

Transgenic plants were evaluated in small field plots at the UF-IFAS Plant Research and Education Center in Citra, Florida, under USDA permit 05-364-01r in a randomized block design with a total of 24 replications. In preparation for the field establishment, transgenic lines H1 and H3 and wild type were propagated under greenhouse conditions from single rooted tillers in 8 × 8 × 7-cm pots with steam sterilized topsoil and 96 replications. For establishment of 1 × 1-m plots, each plot received four transplants, previously grown in of 8 × 8 × 7-cm pots into the corners of the 30 × 30-cm centre on 11 July 2006. The soil type in Citra is Arredondo fine sand with 1% organic matter. All plots were treated equally and irrigated after transplanting to prevent severe moisture stress. Plants were fertilized with 0.23 kg of N per 100 m2 using a complete 18-3-18 NPK fertilizer on 26 July 2006. Prowl (Pendimethalin) herbicide was applied as a pre-emergent to all plots at 7.4 × 10-3 kg per 100 m2 on 1 August 2006. After transplanting plants were allowed to grow without being mowed for 4 weeks. Plants were mowed the second time on 3 October 2006, when a weekly mowing regime was established. Above ground biomass was harvested at 8-cm cutting height to evaluate biomass production using a Rotary Mower HRX217TDA (American Honda Motor, Alpharetta, GA, USA). Clippings were collected from each individual plot and the dry weight of the clippings was assessed after drying at 80 °C. Turf density was evaluated by counting number of tillers in a randomly selected 10 × 10 cm area of each plot. The number of inflorescences per plot and the length of the inflorescence stems were also evaluated. During flowering racemes were cut twice a week at the end of the reproductive stem to prevent dispersal of pollen and prevent formation of seeds. Summer solstice was on 21 June 2006 with a day length of 14 h. National Turfgrass Evaluation Program visual ratings of turf quality were also taken. Visual ratings were based on a 1-9 rating scale, 1 representing the poorest and 9 the best overall quality. Turf quality was rated following mowing 14 weeks after plot establishment of field plots and considered steminess, gaps and aesthetic appearance after mowing.

Statistical analysis

Statistical analysis was performed according to the randomization structure using the analysis of variance of SAS version 9.1 (SAS Institute). Means were compared by the t-test (LSD, P < 0.05). Standard error is shown in figures as vertical bar.

Acknowledgements

We thank Thomas Sinclair, Agronomy Department, University of Florida-IFAS for helpful discussions and critical reading of the manuscript, Anja Liebrandt for skilled technical assistance with gibberellin extraction and analysis and Jeff Seib for training in safe handling of radioisotopes. This research was funded in part by grants from the Consortium for Plant Biotechnology Research, the Scotts Company and the Southwest Florida Water Management District.

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