J. Plant Physiol. 160. 945 952 (2003) Urban & Fischer Verlag http://www.urbanfischer.

de/journals/jpp

Transformation of peanut using a modified bacterial mercuric ion reductase gene driven by an actin promoter from Arabidopsis thaliana
Hongyu Yang1, Joe Nairn2, Peggy Ozias-Akins1 *
1 2

Department of Horticulture, University of Georgia Tifton Campus, Tifton, GA 31793-0748, USA School of Forest Resources, University of Georgia, Athens, GA 30602, USA

Received October 9, 2002 Accepted February 7, 2003

Summary
In order to test an alternative selectable marker system for the production of transgenic peanut plants (Arachis hypogaea), the bacterial mercuric ion reductase gene, merA, was introduced into embryogenic cultures via microprojectile bombardment. MerA reduces toxic Hg(II) to the volatile and less toxic metallic mercury molecule, Hg(0), and renders its source Gram-negative bacterium mercury resistant. A codon-modified version of the merA gene, MerApe9, was cloned into a plant expression cassette containing the ACT2 promoter from Arabidopsis thaliana and the NOS terminator. The expression cassette also was inserted into a second vector containing the hygromycin resistance gene driven by the UBI3 promoter from potato. Stable transgenic plants were recovered through hygromycin-based selection from somatic embryo tissues bombarded with the plasmid containing both genes. However, no transgenic somatic embryos were recovered from selection on 50100 mol/L HgCl2. Expression of merA as mRNA was detected by Northern blot analysis in leaf tissues of transgenic peanut, but not in somatic embryos. Western blot analysis showed the production of the mercuric ion reductase protein in leaf tissues. Differential responses to HgCl2 of embryo-derived explants from segregating R1 seeds of one transgenic line also were observed. Key words: Arachis hypogaea groundnut selectable marker Abbreviations: ACT2 = actin-2. EIM = embryo induction medium. EM = embryogenesis medium. hph = hygromycin phosphotransferase. IPT = isopentenyl transferase. merA = mercuric ion reductase. MS = Murashige and Skoog. PMI = phosphomannose isomerase. PVPP = polyvinylpolypyrollidone

Introduction
The production of transgenic plants with novel traits has relied largely on the use of selectable marker genes conferr* E-mail corresponding author: ozias@tifton.uga.edu

ing antibiotic or herbicide resistance. Thus far, most peanut transformation systems have used hygromycin phosphotransferase (hph), a gene that confers resistance to the antibiotic hygromycin, as the selectable marker gene (Singsit et al. 1997, Wang et al. 1998, Yang et al. 1998, Livingstone and Birch 1999, Magbanua et al. 2000, Ozias-Akins and Gill 2001).
0176-1617/03/160/08-945 $ 15.00/0

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cultivar, Georgia Runner. Mature seeds were disinfected by shaking for one hour in 20 % Clorox, followed by rinsing several times in sterile deionized water. Embryo axes were excised and cultured on embryo induction medium (EIM) containing MS salts (Murashige and Skoog 1962), B5 vitamins (Gamborg et al. 1968), 30 g/L sucrose, 3 mg/L picloram (Ozias-Akins et al. 1993) and solidified with 8 g/L agar (Gum Agar, Sigma, St. Louis, MO). The pH was adjusted to 5.8 prior to autoclaving. The initiation of embryogenic cultures on EIM occurred within 3 weeks in the dark at 28 C. After the initiation phase, all cultures were maintained on embryogenesis medium (EM) with a 2 3 week subculture interval. The EM was identical to EIM except that the latter also contained 1 g/L glutamine (filter-sterilized). Bombardments were conducted on cultures two weeks after subculture.

However, the use of antibiotic resistance genes in transgenic peanuts complicates the commercialization of such lines because of unfavorable public perception and freedom to operate issues. Ecological concerns have been raised regarding the transfer of herbicide resistance genes used as selectable markers through cross-pollination to congeners, some of which may be weeds. Therefore, a selection system for plant transformation that does not require the use of antibiotics or herbicides is desirable. In recent years several alternative selectable markers have been tested including isopentenyl transferase (IPT) from A. tumefaciens (Ebinuma et al. 1997, Sugita et al. 1999, Endo et al. 2001), xylose isomerase (Haldrup et al. 1998), phosphomannose isomerase (PMI) (Joersbo et al. 1998, Zhang et al. 2000, Lucca et al. 2001, Reed et al. 2001, Wright et al. 2001) and cyanobacterial GR6 glutamate-1-semialdehyde aminotransferase (Gough et al. 2001). Phosphomannose isomerase may not be suitable for selection in legumes since they have been reported to contain endogenous PMI activity (Lee and Matheson 1984). As with antibiotic resistance genes, mercury resistance genes are commonly found in bacteria, including E. coli, and are most frequently plasmid borne (Summers 1986). Pike et al. (2002) showed that a high percentage of children ( >70 %) harbor oral bacteria that are mercury resistant. Horizontal transfer of mercury resistance genes can occur among bacteria (Mindlin et al. 2002), but it has been shown that horizontal gene transfer from plants to microorganisms is rare (Thomson 2001), and the use of an already pervasive mercury resistance trait as a selectable marker in plants should pose no threat to human health. A modified bacterial gene encoding mercuric ion reductase (merA), which reduces highly toxic, ionic mercury Hg(II) to a volatile, much less active, elemental form, Hg(0) (Fox and Walsh 1982), has been transferred to yellow poplar (Rugh et al. 1998) for the purpose of phytoremediation of heavily contaminated soils. For poplar, embryogenic cultures were bombarded with plasmids harboring the mercuric ion reductase (merA) gene and the NPTII gene for kanamycin resistance. Transformants were selected on kanamycin and subsequently assayed on mercury-containing medium. Most of the kanamycin-resistant lines were also resistant to 50 mol/L HgCl2, and were capable of developing into mature somatic embryos under selective pressure. In this paper we investigated the characteristics of mercuric ion metabolism in transgenic peanut plants transformed with a codon-modified merA gene and tested the suitability of merA as a selectable marker gene for peanut transformation.

Construction of transformation vectors

In order to test the suitability of the merA gene as an alternative selectable marker for peanut transformation, plasmids pAC2MR and pACH2MR were constructed. The plasmids differed in the presence or absence of the selectable marker gene for hygromycin resistance, hph. In pAC2MR, the mercuric ion reductase gene merApe9 (Rugh et al. 1996) was inserted as a 1.7 kb BamHI/HindIII fragment into the multiple cloning site of pAPC-III. This vector is a pUC-based plasmid into which the actin-2 (ACT2) promoter from Arabidopsis thaliana (the ACT2 promoter was obtained from R. B. Meagher, University of Georgia and modified from the ACT2 GUS construct of An et al. 1996) was inserted and separated by a multiple cloning site from the NOS terminator. The choice of the Arabidopsis promoter, ACT2, to drive merA, was based on expression data from An et al. (1996), who showed it to be largely constitutive. To construct pACH2MR, the merA gene cassette was excised from pAC2MR with two flanking, rare-cutting enzymes, SpeI and AscI, and ligated into pAPCH-III. This vector contains the hph gene under the control of the UBI3 promoter from potato (Garbarino and Belknap 1994) and the NOS terminator.

Microprojectile bombardment
E. coli containing the transformation vectors was grown overnight in LB medium supplemented with ampicillin (100 mg/L). Plasmid DNAs for bombardment experiments were isolated using the QIAGEN Plasmid Maxi/Midi Kit (Qiagen Inc, Valencia, CA). All bombardments were conducted with the Biolistic PDS 1000/He system (Bio-Rad, Hercules, CA) and somatic embryos two weeks after subculture. Twenty clusters of somatic embryos, with 3 to 5 embryos per cluster, were gathered in the central 2-cm area of the Petri dish. Thirty minutes prior to particle bombardment, tissues were desiccated by uncovering the plates and exposing them to airflow in the laminar flow hood. For microcarrier preparation, gold particles (60 mg) of 1.0-m diameter (Bio-Rad) were washed twice with 100 % ethanol, twice with sterile distilled water and resuspended in 1 mL water in a 1.5 mL microcentrifuge tube (United Scientific Products, San Leandro, CA). Plasmid DNA (6 g), 50 L 2.5 mol/L CaCl2, and 20 L 0.1 mol/L spermidine were added to 50 L of gold particle suspension. The mixture was vortexed for 5 min, pelleted by a brief spin, washed once with 250 L of 100 % ethanol, and resuspended in 70 L of 100 % ethanol. An aliquot (10 L or 0.5 mg) of resuspended, DNA-coated gold particles was distributed onto each macrocarrier and used for the bombardment of one plate. Somatic embryo tissues were bombarded at 12,410 kPa (1800 psi) and 91 kPa (27 in of Hg) vacuum. Bombardment conditions were as de-

Materials and Methods

Plant materials and media
Embryogenic cultures of peanut (Arachis hypogaea L.) were initiated from embryo axes of mature seeds (McKently 1991) of the commercial

Mercury resistance in peanut

scribed by Ozias-Akins et al. (1993). The macrocarrier travel distance was 16 mm, and the microcarrier travel distance was 6 cm.

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Selection and regeneration of putatively transformed cell lines

The response of somatic embryo tissues to different dosages of mercury (HgCl2) was tested by plating non-transformed peanut somatic embryos onto EM culture medium containing 0, 50, 100 and 200 mol/ L HgCl2. Three plates were cultured for each treatment, and each plate contained 15 embryogenic clusters with 2 4 embryos per cluster and a total fresh weight of 150 to 200 mg. The culture conditions for the test were the same as for embryo maintenance. Four weeks after transfer to mercury-containing medium, the number of embryos present on each embryogenic tissue piece and the weight of tissues on each plate were recorded. Data were subjected to ANOVA using SAS (SAS Institute, Inc. 2000). After bombardment, somatic embryos were incubated on the same plate for 2 d in the dark at 25 C. Then each plate was transferred to a 250 mL Erlenmeyer flask containing 25 mL liquid EM medium supplemented with either 50 mol/L HgCl2 for the merA-containing plasmid or 20 mg/L hygromycin for the merA + hph-containing plasmid. Flasks were shaken continuously at 130 rpm in the dark at 26 C. The liquid medium was withdrawn and replaced every two weeks allowing either continuous selection with hygromycin or alternating selection/no selection for HgCl2. Two months after bombardment, most tissues exposed to the selection agents ceased growth and became necrotic. Tissues that showed fresh growth were removed from liquid medium and transferred to semi-solid medium containing the selection agent. Only tissues that were capable of proliferation under selection were considered to be putative transgenic lines. Plant regeneration and rooting were carried out according to Ozias-Akins (1989) and OziasAkins et al. (1993).

Taq DNA polymerase (Promega, Madison, WI). PCR was performed in a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT) using the following cycling conditions: initial denaturation at 94 C for 10 min followed by 40 cycles of amplification at 94 C for 30 s, 55 C for 30 s, and 72 C for 1 min. After the final cycle, a 10-min extension at 72 C was added. Southern blotting and hybridization. Cell lines and plants that showed a positive PCR signal for the presence of transgenic DNA were subjected to Southern blot analysis. Two restriction enzymes, HindIII or BamHI, were separately used to digest genomic DNA for two different blots. Only one cut site for HindIII is present in the plasmids. Fragments were separated by electrophoresis on a 0.8 % agarose gel in 1 TBE. DNA was transferred to GeneScreen Plus nylon membrane (NEN Research Products, Boston, MA) using 0.4 N NaOH following the downward transfer protocol (Chomczynski 1992). A 900 bp gene-specific probe for merA was labeled with [-32P]dCTP by PCR following the procedure of Schowalter and Sommer (1989) and using the primers MerS and 900A described above. Pre-hybridization and hybridization were carried out at 65 C in a solution containing 7% SDS, 0.25 mol/L phosphate buffer, 1mmol/L EDTA, and 1% bovine serum albumin (Church and Gilbert 1984). The most stringent wash was carried out in 0.1 SSC and 0.5 % SDS at 65 C. After overnight exposure to a storage phosphor screen, the signals were read by the Cyclone Imaging System with OptiQuant software (Packard, Meriden, CT).

Analysis of transgene expression

Northern blots. Total RNA was isolated from young leaves according to Knapp and Chandle (1996). Prior to electrophoresis, 10 g RNA was denatured by heating to 65 C for 15 min in 10 L formamide, 2 L of 10 MOPS buffer, and 3.5 L of formaldehyde in a total volume of 30 L. Samples were fractionated through a 1 % agarose gel containing 1 MOPS (40 mmol/L MOPS, 1 mmol/L EDTA, 10 mmol/L sodium acetate) and 20 % formaldehyde using 1 MOPS running buffer without circulation. The gel stained with ethidium bromide was used to adjust for variation in RNA loading. RNA was transferred to GeneScreen Plus nylon membrane and hybridized to a PCR-labeled DNA probe specific for the merA gene. Western blot analysis. MerA expression in transgenic peanut lines was examined by Western blot analysis according to the protocol described in Rugh et al. (1996) with minor modifications. Crude protein extracts prepared from leaf tissues of peanut plants growing in the greenhouse were separated by SDS-PAGE and electro-transferred to Immuno-BlotTM PVDF-membrane (Bio-Rad Laboratories, Hercules, CA) using a Mini Trans-Blot Electrophoresis Transfer Cell (Bio-Rad) following the manufacturers recommendations. The blots were probed with a monoclonal anti-MerA antibody produced by the University of Georgia Monoclonal Antibody Facility (Athens, GA). Detection of the MerA antibody was carried out with a secondary antimouse IgG conjugated with horseradish peroxidase (Amersham Pharmacia Biotech, Piscataway, NJ) and a horseradish peroxidase-based Enhanced Chemoluminescent Detection System (Amersham Pharmacia Biotech, Piscataway, NJ). Mercury vapor assay. Individual transgenic plants were analyzed by an in vitro enzyme assay that detected the volatile product Hg(0) of the mercuric ion reductase. Volatized Hg(0) was measured on a Jerome 431 mercury vapor analyzer (Arizona Instrument, Tempe, AZ) according to the protocol of Rugh et al. (1996) with modifications. Two to three leaves, approximately 70 80 mg total wet weight, were

DNA analysis
DNA Extraction. Genomic DNA was extracted from embryogenic or leaf tissues using a modification of the CTAB protocol described by Murray and Thompson (1980). Briefly, 70100 mg of young tissue were ground with a plastic pestle in a 1.5 mL microcentrifuge tube containing 0.6 mL of extraction buffer [2 % w/v CTAB, 1.4 mol/L NaCl, 20 mmol/L EDTA, 100 mmol/L Tris. HCl, pH 8.0, 1 % (w/v) insoluble polyvinylpolypyrollidone (PVPP, Sigma) and 0.2 % -mercaptoethanol] at room temperature. Samples were incubated at 65 C for 15 min with occasional shaking, then extracted twice with an equal volume of chloroform-isoamyl alcohol (24 : 1). Ice-cold isopropanol (0.6 vol) was added to the aqueous phase and genomic DNA was collected by centrifugation at 13,000 g for 10 min. Genomic DNA was resuspended in 250 L H2O. RNA was removed by adding 5 L of a stock solution of 2 mg/mL RNAse A and 5000 U/mL RNAse T1 and incubating the samples at 37C for 30 min. PCR. Two primers, MerS (5-ATG AGC ACT CTC AAA ATC AC-3 and 900A (5-GCG TGC AAG AAT GGT CAC TT-3), were used for the screening of the putative transformants for the merA transgene. The primer pair produced a PCR product of 900 bp which comprised part of the coding region of the merA gene. Generally, 100 ng template DNA was used in a 25 L PCR reaction containing 1 reaction buffer (50 mol/L KCl, 10 mmol/L Tris-HCl, pH 9.0, 0.1 % Triton X-100 and 2.5 mmol/L MgCl2), 100 mmol/L dNTPs, 5 pmol of each primer, and 1U

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L BAP, and 2 mg/L 2,4-D (Sharma and Anjaiah 2000). Cultures initially were incubated for 14 d at 26 C under a 16 h photoperiod with a light intensity of approximately 40 mol m 2 s 1. Subculture was carried out under the same conditions of temperature and light on HgCl2-containing (100 mol/L) shoot-elongation medium composed of MS salts, B5 vitamins, 1.0 mg/L BAP and 0.2 mg/L NAA. Growth and shoot regeneration were evaluated after three weeks.

soaked in 2 mL of assay medium (50 mmol/L Tris-HCl, pH 6.8, 50 mmol/L NaCl) in a 16 130 mm test tube. The reaction was initiated by adding HgCl2 to a final concentration of 25 mol/L. A time-zero reading was taken immediately after the leaf tissues were suspended in the solution, and subsequent readings were taken at 1-min intervals. The release of volatized Hg(0) was facilitated by bubbling air through the assay solution for 12 s at a rate of 3 cm3/s. The relative elemental mercury release values were obtained by dividing the amount (ng) of Hg(0) measured by the mass (mg) of leaf tissues used in the assay. There were three replications of each treatment. Data were subjected to statistical analysis of variance using SAS (SAS Institute, Inc 2000).

Results
Mercury toxicity to peanut somatic embryos
Increasing concentrations of HgCl2 in the culture medium led to a significant inhibition of proliferation and growth of nontransformed somatic embryos (Table 1). All mercury treatments showed a significantly smaller weight than controls (P < 0.01). After four weeks on medium with 100 and 200 mol/ L HgCl2 most tissues remained white. There was, however, no sign of growth or production of new embryos. The concentration of 50 mol/L HgCl2 caused browning of some tissues and greatly reduced the growth of non-transformed tissues but still allowed a few new embryos to emerge. At 50 mol/L HgCl2, an average of only 1.6 embryos per cluster was produced, while the controls produced approximately 10 embryos per cluster. Subsequent selection for mercury resistance was carried out at 50 mol/L HgCl2 because this level of mercury was sufficient to greatly retard the growth of nontransformed tissues but often did not kill them immediately.

Test of mercury selection during organogenesis

Explants from mature seeds harvested from transgenic peanut line X73-5-1 were prepared according to Li et al. (1994). Briefly, peanut seeds were collected from greenhouse-grown peanut plants, surfacesterilized in 20 % Clorox for 30 min with shaking at 120 rpm, then rinsed three times in sterile, deionized water. The seed coats were removed and the seeds were separated into two parts. One part consisted of one cotyledon which was saved for DNA analysis (to determine the presence of the transgene). The other part constituted the second cotyledon with attached embryo axis from which the embryonic radicle and shoot apex were removed. Explants were incubated on shoot induction medium composed of MS salts, B5 vitamins, 5 mg/

Table 1. Effect of HgCl2 on growth and proliferation of non-transformed peanut somatic embryos after 4 weeks of exposure. Means followed by the same letter are not significantly different (P < 0.01). Treatment (mol/L) 0 50 100 200 Average number of new embryos/cluster 9.8 A 1.6 B 0C 0C Average weight (mg/plate) 1162.3 A 398.3 B 309.3 B 153.0 C

Recovery of merA-expressing transgenic peanut plants by bombardment with plasmid pACH2MR

The plasmid pACH2MR, containing the merA gene driven by the ACT2 promoter plus the hph gene controlled by the UBI3 promoter, facilitated the recovery of mercury-resistant plants by selection with the antibiotic hygromycin. Twenty-two hygromycin-resistant lines were recovered from embryos bom-

Figure 1. Southern blot of peanut plants transformed with the mercury resistance gene. Genomic DNAs from peanut and plasmid DNAs were digested with HindIII (A) or BamHI (B) and hybridized with a PCR-probe amplified from pACH2MR. Lane 1: X73-3-3; Lane 2: X73-4-1; Lane 3: X735-1; Lane 4: Georgia Runner; Lane 5 and 6: pACH2MR; Lane 7: 1Kb ladder.

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Figure 2. Northern blot analysis of peanut embryos (lanes 1, 3, 5, 7) and leaf tissues (lanes 2, 4, 6, 8) probed with the PCR-amplified merA fragment from pACH2MR labeled with 32P (A) and the ethidium bromide-stained gel (B). Hybridizing band is 1.9 kb in size. Lanes 1 and 2: X73-3-3; Lanes 3 and 4: X73-4-1; Lanes 5 and 6: X73-5-1; Lanes 7 and 8: Georgia Runner.

browning of embryogenic tissues and no new somatic embryo development. Since the merA gene was driven by the Arabidopsis thaliana ACT2 promoter, which had not been previously tested in stably transformed peanut tissues, the possibility existed that gene expression in embryogenic tissues was insufficient to confer mercury resistance. Expression at the RNA level, therefore, was tested by Northern blot analysis of both leaf and embryogenic tissues (Fig. 2 A). Leaf tissues of X73-4-1 and X73-5-1 showed transcripts of the merA gene in two experiments. No expression was detected in leaf tissues from X73-3-3, even though ethidium bromidestained gels showed adequate RNA loading in this lane (Fig. 2 B). Only weak expression could be observed in embryogenic tissues from X73-4-1 and X73-5-1, thus expression of the ACT2 merA gene appeared to be differentially regulated in the two organ/tissue types. Low expression in embryogenic tissues likely accounted for the failure of transgenic somatic embryos to grow on mercury-containing medium. This observation also could explain our inability to select mercury resistant tissues after bombardment with plasmid pAC2MR where the merA gene also was driven by the ACT2 promoter (data not shown).

barded with pACH2MR through antibiotic selection. Since the merA gene was linked to the hygromycin resistance gene on pACH2MR, we expected that both genes would be co-transferred at a high frequency. The presence of the merA gene was verified in these lines by PCR (data not shown). Out of the 22 transgenic lines, 7 were regenerated to whole plants and the 3 most vigorous (X73-3-3, X73-4-1 and X73-5-1) were tested for the merA gene by PCR and Southern blot analyses. The expected 900 bp DNA fragment was amplified using the merA gene-specific primers with genomic DNA from the transgenic lines but not from the control genotype (non-transgenic Georgia Runner) (data not shown). Integration of the merA gene into genomic DNA was shown by Southern blot analysis (Fig. 1). Different integration patterns and copy number of the merA gene were observed in the three transgenic plants. X73-4-1 (Fig. 1, lane 2) had a relatively simple hybridization pattern and lower copy number, while multiple hybridizing bands were observed for X73-3-3 (lane 1) and X73-5-1 (lane 3). Since HindIII cut only once in the plasmid and the hybridization probe was from the coding region of the merA gene, any bands shown on the blot should represent multiple insertion sites or rearrangements within a site of transgene integration. Plant tissues shown to contain the merA gene presented the best materials for testing the suitability of mercury as a selection agent for peanut transformation. In order to test for mercury resistance, somatic embryos from hygromycin-resistant transgenic lines were transferred to medium containing 50 mol/L HgCl2. None of the transgenic lines showed resistance to mercury and responded after three weeks with

Response of transgenic merA plants to mercury

Plants from the three transgenic lines were tested for resistance to mercury. Shoots from the transgenic plants X73-3-3, X73-4-1 and X73-5-1 and control Georgia Runner were placed on rooting medium containing 100 mol/L HgCl2. The peanut shoots showed a very rapid response to mercury; leaves of non-transgenic plants began to wilt two days after culture initiation and were largely senescent within five days (Fig. 3 A). Leaf discolorations and wilting appeared on the older leaves first. No roots were produced on control plants for the first four weeks, and only a few roots emerged after prolonged culture of 4 to 6 weeks. Those roots were greatly retarded in their growth. Transgenic plants, on the other hand, remained green throughout the rooting process, and root initiation was observed 10 d after culture initiation. The roots grew vigorously and showed no difference in number or length compared with those of transgenic plants growing on medium without mercury (data not shown). The level of mercuric ion reduction was measured in leaf tissues of X73-3-3, X73-4-1, X73-5-1, and Georgia Runner. In vitro-grown leaflets were incubated in liquid medium containing 25 mol/L HgCl2 in order to measure mercury evolution. The assay result showed a clear correlation between mercury resistance and volatile Hg(0) release rates. Elemental mercury evolution rates in mercury-resistant plants were higher than in non-transgenic control plants (Fig. 4). The mercuryresistant lines X73-4-1 and X73-5-1 had a release rate 4 to 6 times higher than Georgia Runner and were significantly different from the control (P < 0.01), while the line X73-3-3 that failed the challenge of 100 mol/L HgCl2 during rooting

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Figure 3. A) Test for resistance to mercury during rooting of somatic embryo-derived shoots. Left: Transgenic; right: Non-transgenic. B C) Regeneration response of cotyledon/mesocotyl explants from T1 seeds of X73-5-1 on HgCl2. B-left: T1 8; B-right: T1 5. C-left: T1 4; C-right: T111.

Figure 4. Elemental mercury [Hg(0)] release assays in transgenic peanut plants. In vitro-grown leaflets of independent transgenic lines were incubated in liquid medium containing 25 mol/L HgCl2. Volatilized Hg(0) was measured on a Jerome 431 mercury vapor analyzer each minute for 10 minutes. There were three replications of each treatment, and data were subjected to ANOVA using SAS. X73-3-3 was not significantly different from the control, whereas X73-4-1 and X73-5-1 were (P < 0.01).

Figure 5. Western blot analysis of MerA protein in transgenic peanut plants. Total protein was extracted from young unfolded leaves, separated by SDS-PAGE and transferred to a PVDF-membrane. The MerA protein immobilized on the membrane was then detected using a monoclonal antibody. Lanes 1 and 6: protein isolated from E. coli expressing MerA protein. Lane 2: X73-3-3; Lane 3: X73-4-1; Lane 4: X73-5-1; Lane 5: Georgia Runner.

Western blot analysis showed a correlation between mercury resistance and the expression of mercuric ion reductase in the leaf tissues. The line X73-3-3 that did not grow on mercury-containing medium showed no MerA protein expression, while the lines X73-4-1 and X73-5-1 that survived the challenge of 100 mol/L of HgCl2 during rooting showed antibody recognition of a single band (Fig. 5). Proteins from the control plant of Georgia Runner did not show any reaction with the MerA antibody. The two bands present in the bacterial control lanes are consistent with previous observations of a presumed proteolytic cleavage product that did not affect activity (Fox and Walsh 1982). Since only the smaller of the two bands was detected in transgenic plants, it is likely that processing is occurring in the peanut leaves.

showed only a slightly higher measurement of Hg(0) than the control. The difference between X73-3-3 and the control was, however, not statistically significant when analyzed by ANOVA. These results strongly supported that the mercury resistant phenotype of the transgenic peanut plants was due to the conversion of mercury from a toxic ionic form to a non- or less-toxic elemental form.

Test of merA as a potential selectable marker gene for transformation in an organogenic system
The results of Northern blot, Western blot, and mercury vapor assays clearly showed that the merA gene was expressed at

Mercury resistance in peanut both the mRNA and protein levels in two transgenic lines and that the mercuric ion reductase enzyme encoded by the merA gene was functional in transforming the toxic mercuric ion to a less toxic elemental form. In order to determine whether merA could be used as a selectable marker gene for an organogenesis-mediated transformation system, T1 seed explants were tested on HgCl2-containing medium as described in Materials and Methods. Of the four seeds tested, two were PCR-positive for the transgene and the other two were negative (data not shown). Cotyledon/mesocotyl explants containing the transgene remained green and produced new shoots while those that did not contain the transgene turned brown and died (Fig. 3 B C).

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Discussion
In an effort to explore the development of an alternative selectable marker for peanut transformation, we have studied the expression of a codon-modified bacterial merA gene under the control of a plant regulatory DNA sequence, the Arabidopsis promoter, ACT2. Although in retrospect, our choice of promoters did not allow us to use merA as a selectable marker gene in embryogenic tissues, it did provide data on the expression pattern that can be expected in peanut and the utility of both the promoter and selectable marker gene during stages of regeneration. Differential expression of actin genes has been reported in several plant species (McElroy et al. 1990, Thangavelu et al. 1993), and such a gene family should provide a useful source of regulatory sequences for transgene expression. In our work, the ACT2 promoter was derived from the Arabidopsis thaliana actin 2 gene that is one member of the large, divergent actin-encoding gene family in higher plants (An et al. 1996). In Arabidopsis, ACT2 is from the vegetative subclass of actin genes and showed strong and constitutive expression in vegetative tissues of leaves, roots, stems and little or no expression in seed coats, hypocotyls, gynoecia and pollen sacs (An et al. 1996). Cotyledons and radicals showed strong expression after, but not prior to germination of seeds. A member of the reproductive subclass, ACT11, was found to be strongly expressed in tissues of the emerging inflorescence, pollen, and developing ovules (Huang et al. 1997). Our results with transgenic plants containing a merA gene driven by the ACT2 promoter demonstrated that merA could be strongly expressed in peanut vegetative tissues, but not in somatic embryos. This pattern, therefore, rendered the ACT2 promoter an unsuitable candidate for controlling selectable marker genes in embryonic stages of development. It recently has been shown that ACT7 is the only Arabidopsis gene that is strongly induced by auxin treatment (Kandasamy et al. 2001), where ACT2 was slightly down-regulated; therefore, ACT7 might provide a more suitable promoter for a selectable marker gene to be expressed in auxin (picloram)induced somatic embryos of peanut. However, because the

merA gene driven by the ACT2 promoter was strongly expressed in leaves of regenerated peanut plants, as was shown in the Northern and Western blot analyses, the ACT2 promoter could reasonably be employed as a regulatory element for marker genes in a transformation system where selection takes place post-embryonically or during organogenesis. Such Agrobacterium-mediated transformation systems that utilize organogenesis have been reported for peanut, although they typically are highly genotype dependent (Eapen and George 1994, Cheng et al. 1996, Sharma and Anjaiah 2000). The use of merA as a selectable marker gene for a transformation system via embryogenesis would require the identification of a suitable promoter. Candidate promoters might be Arabidopsis thaliana ACT7 which is induced in response to exogenous auxin (Kandasamy et al. 2001), ACT11 which drives expression in developing embryos (Huang et al. 1997), or the potato ubiquitin promoter UBI3 (Garbarino et al. 1992) which we have successfully used in this study to drive the hph gene for hygromycin selection.
Acknowledgements. Support for this work was provided by the USDA Multicrop Aflatoxin Elimination Program and the National Peanut Foundation. We thank Evelyn Perry and Anne Bell for technical assistance and Rich Meagher for providing promoters and expertise/facilities for mercury vapor analysis.

References
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