Crop Science 42:1298-1302 (2002)
© 2002 Crop Science Society of America
CELL BIOLOGY & MOLECULAR GENETICS
Transgenic Dry Bean Tolerant to the Herbicide Glufosinate Ammonium
Francisco J. L. Aragão*,
Giovanni R. Vianna,
Margareth M. C. Albino and
Elíbio L. Rech
Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica, Final Av. W3 Norte, 70770-900 - Brasília-DF, Brazil
* Corresponding author (aragao{at}cenargen.embrapa.br)
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ABSTRACT
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The bar gene from Streptomyces hygroscopicus (Jansen) Labeda & Lyons encodes phosphinothricin acetyl transferase (PAT), which confers tolerance to the herbicide glufosinate ammonium. In Brazil, the productivity of Phaseolus vulgaris L. (dry bean) has been declining in some regions. One of most limiting factors is weed competition. This study was conducted to evaluate the possibility of obtaining transgenic bean plants resistant to glufosinate ammonium, which would facilitate weed control during the summer season. The biolistic process was used to insert this gene into dry bean, and the integration was confirmed by Southern blot analysis. Two transgenic events, PHV119 and PHV122 were tolerant to 500 g ha-1 of glufosinate ammonium under green house conditions, with no visible symptoms and developmental growth comparable to nontransgenic P. vulgaris. Field evaluation carried out with the PHV119 event has shown that the plants tolerated up to 400 g GA ha-1, with no visible symptoms. Inheritance studies showed that the transgenes segregated in a Mendelian fashion.
Abbreviations: bp, base pair(s) • GA, glufosinate ammonium • PAT, phosphinothricin acetyl transferase • PCR, polymerase chain reaction • PPT, phosphinothricin
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INTRODUCTION
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IN BRAZIL, dry bean is nutritionally important because it is part of the staple diet, but productivity of this crop has been declining in some regions, with the average Brazilian yield being 600 kg ha-1. This crop has the potential to yield over 4000 kg ha-1, with the West Asian and North American yields being 1100 to 1500 kg ha-1, while yields in Latin American and African countries are between 500 to 600 kg ha-1. In low-yield regions, the main limiting factors are poor agronomic practices, diseases, insects, nutritional deficiencies, soil-type, climatic constraints, lack of improved varieties, and weed competition.
Recombinant DNA technology, associated with traditional sexual breeding methods, may accelerate the production of plants with useful traits. Indeed, the improvement of P. vulgaris through genetic engineering has become a reality (Russel et al., 1993; Aragão et al., 1996, 1998, 1999). Recently, we obtained transgenic events resistant to the Bean golden mosaic virus. To generate virus-resistant cultivars, these events were incorporated into the bean breeding programs.
The presence of weeds in crop fields is an important productivity constraint in tropical regions and an obstacle to the cultivation and harvesting of P. vulgaris. Weed control based on the application of herbicides to herbicide-tolerant transgenic crops has been shown to be practical and useful (Brasileiro et al., 1992; Padgette et al., 1995; Enriquez-Obregon et al., 1998; Mohapatra et al., 1999; Carpenter and Gianessi, 2000; Aragão et al., 2000).
Glufosinate ammonium (GA) is a nonselective herbicide that is converted by plants into phytotoxic phosphinothricin (PPT). The bar gene from Streptomyces hygroscopicus encodes phosphinothricin acetyl transferase (PAT) that acetylates the free NH2 group of PPT and prevents GA toxicity (Thompson et al., 1987). The bar gene can thus confer GA tolerance, and we have introduced this gene into P. vulgaris using particle bombardment. The work reported in this paper demonstrates the usefulness of transformation for the development of an agriculturally important herbicide tolerant P. vulgaris.
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MATERIALS AND METHODS
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Plant Transformation, Cultivation, and Treatment
Transgenic P. vulgaris (cultivars Olathe and Carioca) were produced as described by Aragão et al. (1996), from a total of 11 607 embryonic axes (5708 cv. Olathe and 5899 cv. Carioca) in batches of about 250. Briefly, mature seeds were surface sterilized and soaked in distilled water for 16 to 18 h. Then, the embryonic axes were excised from the seeds and the apical meristems were exposed by removing their primary leaves. The embryonic axes were placed with the apical region directed upward in Petri dishes containing basal MS medium (Murashige and Skoog, 1962) immediately before the bombardment. The bombardment was conducted with a high-pressure helium-driven particle acceleration device built in our laboratory. The embryonic axes were cultivated on MS medium containing 44.3 µM 6-benzylaminopurine (BAP) to induce multiple shoot development. The vector used was pB5/35Sbar (supplied by Aventis), which contains the bar gene, driven by the cauliflower mosaic virus (CaMV) 35S promoter, and the bacterial kanamycin resistance gene (neo) (Fig. 1)
. After 3 wk in culture, the bombarded apical meristems (R0 generation) produced elongated shoots which, when they had reached 2 to 4 cm in length and had rooted, were transferred to a plastic pot containing autoclaved soil:vermiculite (1:1) and covered with a plastic bag for 1 wk to acclimatize. To detect integration events related to glufosinate ammonium (GA) tolerance, the 4537 surviving R0 plantlets (2308 cv. Olathe and 2229 cv. Carioca) were sprayed with an aqueous solution (Liberty, Aventis) of glufosinate ammonium at an application rate equivalent to 100 g GA ha-1. The 24 surviving GA-tolerant plantlets (14 cv. Olathe and 10 cv. Carioca) were transferred to 5-m3 plastic pots containing fertilized soil (haplustox) and maintained in a greenhouse [5–30°C, relative humidity (RH) >70%] and allowed to set seed (R1 generation). The R1 generation seed was sown in 5-m3 plastic pots containing fertilized soil and maintained in a greenhouse (25–30°C, RH >70%). When the R1 generation plants produced the first trifoliolate leaves, they were screened for transformation events by spraying with herbicide at an application rate equivalent to 500 g GA ha-1, and the two surviving events of Olathe were designated PHV119 and PHV122. These transformed events were investigated for the presence of the genes by means of chain reaction (PCR). Once transformation was confirmed, the nomenclature was changed from Rn to Tn.
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Fig. 1. Diagram of the plasmid vector (pB5/35Sbar) used for transformation of P. vulgaris (inner circle). The two outer circles indicate the sequences that were introduced into transgenic events PHV119 and PHV122. The position of the primers used in PCR analysis and the probes for Southern blot analyses are also indicated with arrows.
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PCR Analysis
DNA was isolated from leaf disks by the method of Edwards et al. (1991). Each PCR reaction was carried out in 25 µL of reaction mixture containing 10 mM Tris-HCl (pH 8.4), 50 mM KCl, 2 mM MgCl2, 160 µM of each dNTP, 200 nM of each primer, 2U of Taq polymerase (GIBCO BRL), and about 20 ng of genomic DNA. The mixture was overlaid with mineral oil, denatured for 5 min at 95°C in a thermal cycler (MJ Research, Watertown, MA) and amplified for 35 cycles (95°C for 1 min, 55°C for 1 min, 72°C for 1 min) with a final 7 min cycle at 72°C. One half of the reaction mixture was then loaded directly onto a 1% (w/v) agarose gel, stained with ethidium bromide and visualized with UV light. The P2510 and P2958 primer pair (within the bar gene, Table 1) was used to amplify a 448-bp sequence to screen the transgenic plants. Other primer pairs (Table 1 and Fig. 1) were used to determine which sequences of the pB5/35Sbar plasmid had been introduced into the two transgenic events.
Southern Blot Analysis
Genomic DNA was isolated by the methods of Dellaporta et al. (1983) and Southern blotting and hybridization was carried out as previously described (Sambrook et al., 1989). Genomic DNA (15 µg) was digested with HindIII, separated on a 1% agarose gel and transferred to nylon HyBond membrane. Hybridization was carried out with the PCR-generated 448-bp probe for the bar gene (probe a) and the 638-bp probe for the neo gene (probe b) (Fig. 1). The probes were labeled with [
-32P]dCTP (3000 Ci mol-1) with a random primer DNA labeling kit (Pharmacia Biotech) according to the manufacturer's instructions.
Progeny Analysis
T1 generation analysis was carried out by PCR amplification of the bar gene in the self-pollinated plants. Chi-square (
2) analyses, using the correction factor of Yates (Steel and Torrie, 1980), were performed to determine if the observed segregation ratio was consistent with a Mendelian ratio.
Test for GA Tolerance
The P. vulgaris events PHV119 and PHV122 were self-fertilized, and the resulting seeds were germinated in 5-dm3 plastic pots containing fertilized soil and tested for GA tolerance by spraying the plants at the first trifoliolate leaf stage with GA at an application rate equivalent to 500 g GA ha-1.
Field Trial
To evaluate the resistance of the T2 generation of the PHV119 event under field conditions, a preliminary trial was carried out from November 2000 to February 2001 in Distrito Federal, Brazil (Embrapa Recursos Genéticos e Biotecnologia). Each treatment was repeated thee times in blocks randomly distributed. A block consisted of four rows 4.5 m long, 0.5 m apart. Plants were sown side by side within the row (approximately 5 seeds m-1) and sprayed with 0, 400 (commercial dosage), and 800 g GA ha-1 (Liberty, Aventis) at the first trifoliolate leaf stage. The field release was conducted according to the Brazilian law following the recommendations of the Comissão Técnica Nacional de Biossegurança (National Biosafety Commission), authorization number 01200.001359/2000-19.
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RESULTS
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The results of 48 independent transformation attempts are summarized in Table 2. Out of 11 607 bombarded embryonic axes, 4537 R0 generation plantlets were obtained, of which 24 survived to set seed after spraying with herbicide at an application rate equivalent to 100 g GA ha-1. PCR analyses confirmed the presence of the bar gene in all of these plantlets, all of which developed into phenotypically normal plants that were fertile and set pods and seeds. Several plants were chimeric, revealing few branches that showed herbicide-resistance. All these chimeric plants died 5 wk after the herbicide treatment. All tolerant events (putative T1 generation) were analyzed for the presence of the gene by PCR and tested for GA tolerance by spraying the plants at 500 g GA ha-1. Except for four events, PCR analyses confirmed the presence of the bar gene in the T1 generation. After herbicide application, most of the PCR-positive T1 plants showed typical GA toxicity symptoms and died 2 wk after GA application, as did all control (nontransgenic) plants. However, two transgenic events, PHV119 and PHV122, of Olathe were GA-tolerant (Table 2, Fig. 2)
, with no visible symptoms and growth comparable to that of nontransgenic P. vulgaris.
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Table 2. Glufosinate ammonium tolerance. Summary of independent experiments with the circular form of the plasmid vector pB5/35Sbar.
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Fig. 2. Evaluation of PHV119 for glufosinate ammonium tolerance under field conditions. (a) nontransgenic sprayed with 400 g GA ha-1; (b) nonsprayed transgenic plants and (c) sprayed with 400 g GA ha-1 and (d) 800 g GA ha-1. (e) Detail of a transgenic plant sprayed with 400 g GA ha-1 and (f) 800 g GA ha-1. Photographs were taken 2 wk after herbicide treatment.
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Southern blot analysis of genomic DNA isolated from the R0 and R1 transgenic generations, including events PHV119 and PHV122, was carried out to evaluate the integration of the introduced bar and neo genes, and the results showed the presence of the bar sequence in both PHV119 and PHV122 (Fig. 3)
. Since pB5/35Sbar has a unique HindIII restriction site (Fig. 1), Southern blot analysis of the R0 and R1 generations allowed us to confirm plasmid integration as well as to estimate the number of integration sites of the bar and neo genes (Fig. 3) present in the transgenic plants. The integration pattern for the PHV119 event was the same in both generations, with one integration site of the bar gene and none of the neo gene (Fig. 3). In the case of PHV122, there were two integration sites of the bar and neo genes. Southern blot analysis of the T1 generation showed the same pattern for both probes, revealing that the integrated copies segregated independently (Fig. 3) and that the neo and bar genes were linked. DNA isolated from nontransgenic plants did not hybridize either with the bar or neo probes (Fig. 3).
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Fig. 3. Southern blot of putative transformation events in the T0 (A and B) and T1 (C) generations. DNAs were digested with HindIII and probed with an internal fragment of the bar (A and C) and neo (B) genes. A and B: Lane 1 = transgenic event PHV119, Lane 2 = transgenic event PHV122, Lane 3 = nontransgenic plant, Lane 4 = plasmid vector pB5/35Sbar (50 pg). C: Lanes 1 through 3 = T1 plants from event PHV122, Lane 4 = nontransgenic plant, Lanes 5 through 7 = T1 plants from event PHV119, Lane 8 = plasmid vector pB5/35Sbar (50 pg). The size markers are indicated to the left of the figure.
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The To PHV119 and PHV122 were self-fertilized, and their progeny screened by PCR analysis for the presence of the bar gene, with both PHV119 and PHV122 transferring the foreign gene to the T1 generation according to Mendelian fashion. Segregation was 3:1 for PHV119 and 15:1 for PHV122 (Table 3), results that agree with the Southern blot analyses. Plants containing either copy of the bar gene showed higher GA tolerance, indicating that both copies were functional.
PCR analyses with several primer pairs were carried out to determine which sequences were effectively introduced. In PHV119, the plasmid sequence was interrupted between bp 3937 and 637 and bp 870 and 1217, while in PHV122, two sequences of the plasmid were independently integrated, with a gap between bp 1217 and 2100. Southern blot analysis of the PHV122 event revealed the same pattern both for the bar and neo probes (Fig. 1). These results could indicate rearrangement of the plasmid vector during transformation, integration, or stabilization of the insert (De Block, 1993).
Preliminary field trial evaluation carried out with PHV119 has shown that the plants present a high tolerance to 400 g GA ha-1, with no visible symptoms. However, when the plants were sprayed with concentrations of 800 g GA ha-1, herbicide phytotoxicity symptoms were detected, i.e., yellowing of treated leaves. Typically, all nontransgenic plants died 1 wk after application of 400 g GA ha-1 (Fig. 2).
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DISCUSSION
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During the first stage of this experiment, a great number of transgenic and nontransgenic plantlets were obtained because no selective agent was added to the medium during cultivation of the embryonic axes of the R0 generation. In addition, some chimeric plantlets were observed. To reduce the number of plantlets used in PCR analysis to a manageable number, the plantlets were preselected by applying 1/5 of the final herbicide concentration, equivalent to 100 g GA ha-1, to eliminate GA-sensitive plants. Herbicide applications to in vitro emerging shoots in the first stage of the protocol might minimize the number of nontransgenic or chimeric plants.
Of the 24 transgenic R0 events tolerant to the equivalent of 100 g GA ha-1, only two R1 events were tolerant when the GA concentration was increased 5-fold. Since PCR analysis indicated that all 24 events contained at least one copy of the bar gene the resistance level in these two events may be due to lack of sufficient transgene expression. Except for four T0 transgenic events, all events tolerant to 100 g GA ha-1 transferred the transgenes to the T1 generation. The phenomenon of nontransference to the T1 generation was previously observed in transgenic P. vulgaris (Aragão et al., 1996), petunia (Petunia x hybrida Vilm., Ulian et al., 1994) and maize (Zea mays L., Register et al., 1994).
Questions regarding biosafety issues have recently been raised because of the presence of genes for antibiotic resistance in transgenic plants (Dale, 1999), and sequences coding for antibiotic resistance are consequently being avoided for the development of new cultivars. Consequently, we chose the event PHV119 that does not carries the antibiotic resistance gene for a preliminary field trail evaluation. This event behaved similarly under green house and field conditions and has been included in the EMBRAPA Rice and Bean breeding program.
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ACKNOWLEDGMENTS
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The authors are grateful to Josué Ignácio Lemos and Warley Silva Almeida for technical assistance. This study was supported by Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Aventis.
Received for publication March 20, 2001.
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ABSTRACT
INTRODUCTION
