10310069_Yang

7/31/2019 10310069_Yang

http://slidepdf.com/reader/full/10310069yang 1/8

J. Plant Physiol. 160. 945– 952 (2003) Urban & Fischer Verlaghttp://www.urbanfischer.de/journals/jpp

Transformation of peanut using a modified bacterial mercuric ionreductase gene driven by an actin promoter from Arabidopsis thaliana

Hongyu Yang1, Joe Nairn2, Peggy Ozias-Akins1 *

1 Department of Horticulture, University of Georgia Tifton Campus,Tifton, GA 31793-0748, USA2 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 embryo-

genic 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 hyg-romycin-based selection from somatic embryo tissues bombarded with the plasmid containing both

genes. However, no transgenic somatic embryos were recovered from selection on 50–100 µmol/L

HgCl2. Expression of merA as mRNA was detected by Northern blot analysis in leaf tissues of trans-

genic peanut, but not in somatic embryos. Western blot analysis showed the production of the mer-

curic ion reductase protein in leaf tissues. Differential responses to HgCl 2 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 = polyvinyl-

polypyrollidone

Introduction

The production of transgenic plants with novel traits has

relied largely on the use of selectable marker genes conferr-

* E-mail corresponding author: [email protected]

ing antibiotic or herbicide resistance. Thus far, most peanut

transformation systems have used hygromycin phosphotrans-

ferase (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

7/31/2019 10310069_Yang


946 Hongyu Yang, Joe Nairn, Peggy Ozias-Akins

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 systemfor plant transformation that does not require the use of anti-

biotics 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 isome-

rase (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 con-

tain endogenous PMI activity (Lee and Matheson 1984).

As with antibiotic resistance genes, mercury resistance ge-nes 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 bac-

teria (Mindlin et al. 2002), but it has been shown that horizon-

tal gene transfer from plants to microorganisms is rare (Thom-

son 2001), and the use of an already pervasive mercury re-

sistance trait as a selectable marker in plants should pose no

threat to human health.

A modified bacterial gene encoding mercuric ion reduc-

tase (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 con-

taminated soils. For poplar, embryogenic cultures were bom-

barded with plasmids harboring the mercuric ion reductase

(merA) gene and the NPTII gene for kanamycin resistance.

Transformants were selected on kanamycin and sub-

sequently assayed on mercury-containing medium. Most of

the kanamycin-resistant lines were also resistant to 50 µmol/L

HgCl2, and were capable of developing into mature somatic

embryos under selective pressure.

In this paper we investigated the characteristics of mercu-

ric ion metabolism in transgenic peanut plants transformedwith a codon-modified merA gene and tested the suitability of

merA as a selectable marker gene for peanut transformation.

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

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), 30g/L sucrose, 3 mg/L pic-

loram (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 auto-

claving. The initiation of embryogenic cultures on EIM occurred within3 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 1g/L glutamine (filter-sterilized). Bombardments were

conducted on cultures two weeks after subculture.

Construction of transformation vectors

In order to test the suitability of the merA gene as an alternative se-

lectable 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 etal. 1996) was inserted as a 1.7kb Bam HI/ Hin dIII 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 Geor-

gia and modified from the ACT2 ϻGUS 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-cut-

ting enzymes, Spe I and Asc I, 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 Plas-

mid 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-µm diameter (Bio-Rad) werewashed 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 µg), 50 µL

2.5mol/L CaCl2, and 20µL 0.1mol/L spermidine were added to 50µL

of gold particle suspension. The mixture was vortexed for 5 min, pel-

leted by a brief spin, washed once with 250 µL of 100 % ethanol, and

resuspended in 70µL of 100 % ethanol. An aliquot (∼10µL or ∼0.5mg)

of resuspended, DNA-coated gold particles was distributed onto

each macrocarrier and used for the bombardment of one plate. So-

matic embryo tissues were bombarded at 12,410 kPa (1800 psi) and

91kPa (27 in of Hg) vacuum. Bombardment conditions were as de-

7/31/2019 10310069_Yang


947Mercury resistance in peanut

scribed by Ozias-Akins et al. (1993). The macrocarrier travel distance

was 16mm, and the microcarrier travel distance was 6cm.

Selection and regeneration of putatively transformedcell lines

The response of somatic embryo tissues to different dosages of mer-cury (HgCl2) was tested by plating non-transformed peanut somatic

embryos onto EM culture medium containing 0, 50, 100 and 200µmol/

L HgCl2. Three plates were cultured for each treatment, and each

plate contained 15 embryogenic clusters with 2–4 embryos per clus-

ter and a total fresh weight of 150 to 200mg. 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 pre-

sent 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

250mL Erlenmeyer flask containing 25 mL liquid EM medium supple-

mented with either 50 µmol/L HgCl2 for the merA-containing plasmidor 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 se-

lection for HgCl2. Two months after bombardment, most tissues ex-

posed 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 Ozias-

Akins et al. (1993).

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, 70–100mg of young tissue were

ground with a plastic pestle in a 1.5 mL microcentrifuge tube contain-

ing 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% β-mercaptoethanol]

at room temperature. Samples were incubated at 65 ˚C for 15min 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 resus-pended in 250µL H2O. RNA was removed by adding 5 µL of a stock

solution of 2 mg/mL RNAse A and 5000 U/mL RNAse T1 and incubat-

ing the samples at 37˚C for 30min.

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 µL PCR reaction containing 1× reaction buffer

(50 µmol/L KCl, 10 mmol/L Tris-HCl, pH 9.0, 0.1 % Triton X-100 and

2.5 mmol/L MgCl2), 100mmol/L dNTPs, 5 pmol of each primer, and 1U

Taq DNA polymerase (Promega, Madison, WI). PCR was performed in

a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT) using the fol-

lowing cycling conditions: initial denaturation at 94 ˚C for 10min fol-

lowed 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 DNAwere subjected to Southern blot analysis. Two restriction enzymes,

Hin dIII or Bam HI, were separately used to digest genomic DNA for

two different blots. Only one cut site for Hin dIII is present in the plas-

mids. Fragments were separated by electrophoresis on a 0.8 % aga-

rose 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 [α-32P]dCTP

by PCR following the procedure of Schowalter and Sommer (1989)

and using the primers MerS and 900A described above. Pre-hybridi-

zation and hybridization were carried out at 65 ˚C in a solution con-

taining 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 over-night 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 µg RNA

was denatured by heating to 65 ˚C for 15min in 10µL formamide, 2µL

of 10 × MOPS buffer, and 3.5µL of formaldehyde in a total volume of

30µL. Samples were fractionated through a 1% agarose gel contain-

ing 1× MOPS (40 mmol/L MOPS, 1 mmol/L EDTA, 10 mmol/L sodium

acetate) and 20 % formaldehyde using 1× MOPS running buffer with-out circulation. The gel stained with ethidium bromide was used to ad-

just 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 de-

scribed 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 manufacturer’s recommendations. The blots were

probed with a monoclonal anti-MerA antibody produced by the Uni-

versity of Georgia Monoclonal Antibody Facility (Athens, GA). Detec-tion of the MerA antibody was carried out with a secondary anti-

mouse IgG conjugated with horseradish peroxidase (Amersham Phar-

macia Biotech, Piscataway, NJ) and a horseradish peroxidase-based

Enhanced Chemoluminescent Detection System (Amersham Pharma-

cia 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 Je-

rome 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

7/31/2019 10310069_Yang



soaked in 2 mL of assay medium (50 mmol/L Tris-HCl, pH 6.8,

50 mmol/L NaCl) in a 16×130mm test tube. The reaction was initiated

by adding HgCl2 to a final concentration of 25 µmol/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 inter-

vals. The release of volatized Hg(0) was facilitated by bubbling air

through the assay solution for 12s at a rate of 3cm 3 /s. The relative ele-

mental 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).

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, surface-

sterilized in 20 % Clorox for 30 min with shaking at 120 rpm, then

rinsed three times in sterile, deionized water. The seed coats were re-

moved and the seeds were separated into two parts. One part con-sisted of one cotyledon which was saved for DNA analysis (to deter-

mine the presence of the transgene). The other part constituted the

second cotyledon with attached embryo axis from which the embryo-

nic radicle and shoot apex were removed. Explants were incubated

on shoot induction medium composed of MS salts, B5 vitamins, 5mg/

Table 1. Effect of HgCl2 on growth and proliferation of non-trans-

formed peanut somatic embryos after 4 weeks of exposure. Means

followed by the same letter are not significantly different (P <0.01).

Treatment Average number of Average weight

(µmol/L) new embryos/cluster (mg/plate)

0 9.8 A 1162.3 A

50 1.6 B 398.3 B

100 0 C 309.3 B

200 0 C 153.0 C

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 µm o l · m–2 · s–1. Subculture was carried

out under the same conditions of temperature and light on HgCl 2-con-

taining (100µmol/L) shoot-elongation medium composed of MS salts,

B5 vitamins, 1.0 mg/L BAP and 0.2 mg/L NAA. Growth and shoot re-

generation were evaluated after three weeks.

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 non-

transformed somatic embryos (Table 1). All mercury treat-

ments showed a significantly smaller weight than controls

(P <0.01). After four weeks on medium with 100 and 200µmol/

L HgCl2 most tissues remained white. There was, however, no

sign of growth or production of new embryos. The concentra-

tion of 50µmol/L HgCl2 caused browning of some tissues andgreatly reduced the growth of non-transformed tissues but

still allowed a few new embryos to emerge. At 50 µmol/L

HgCl2, an average of only 1.6 embryos per cluster was pro-

duced, while the controls produced approximately 10 em-

bryos per cluster. Subsequent selection for mercury resist-

ance was carried out at 50µmol/L HgCl2 because this level of

mercury was sufficient to greatly retard the growth of non-

transformed tissues but often did not kill them immediately.

Recovery of merA-expressing transgenic peanut plantsby 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 hygro-

mycin-resistant lines were recovered from embryos bom-

Figure1. Southern blot of peanut plants transformed with the mercury resistance gene. Genomic DNAs from peanut and plasmid DNAs were di-

gested with Hin dIII (A) or Bam HI (B) and hybridized with a PCR-probe amplified from pACH2MR. Lane 1: X73-3-3; Lane 2: X73-4-1; Lane 3: X73-

5-1; Lane 4: Georgia Runner; Lane 5 and 6: pACH2MR; Lane 7: 1Kb ladder.

7/31/2019 10310069_Yang



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 bromi-

de-stained gel (B). Hybridizing band is ∼1.9kb 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.

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-trans-

ferred 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-trans-

genic 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 num-

ber of the merA gene were observed in the three transgenic

plants. X73-4-1 (Fig. 1, lane 2) had a relatively simple hybridi-

zation pattern and lower copy number, while multiple hybri-

dizing bands were observed for X73-3-3 (lane 1) and X73-5-1

(lane 3). Since Hin dIII cut only once in the plasmid and the

hybridization probe was from the coding region of the merAgene, any bands shown on the blot should represent multiple

insertion sites or rearrangements within a site of transgene in-

tegration.

Plant tissues shown to contain the merA gene presented

the best materials for testing the suitability of mercury as a se-

lection agent for peanut transformation. In order to test for

mercury resistance, somatic embryos from hygromycin-resist-

ant transgenic lines were transferred to medium containing

50µmol/L HgCl2. None of the transgenic lines showed resist-

ance to mercury and responded after three weeks with

browning of embryogenic tissues and no new somatic em-

bryo 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 tis-

sues was insufficient to confer mercury resistance. Ex-

pression at the RNA level, therefore, was tested by Northernblot 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 bromide-

stained gels showed adequate RNA loading in this lane

(Fig. 2 B). Only weak expression could be observed in em-

bryogenic tissues from X73-4-1 and X73-5-1, thus expression

of the ACT2 ϻmerA gene appeared to be differentially regu-

lated in the two organ/tissue types. Low expression in em-

bryogenic tissues likely accounted for the failure of trans-

genic somatic embryos to grow on mercury-containing me-

dium. This observation also could explain our inability to se-

lect mercury resistant tissues after bombardment with plas-mid pAC2MR where the merA gene also was driven by the

ACT2 promoter (data not shown).

Response of transgenic merA plants to mercury

Plants from the three transgenic lines were tested for resist-

ance 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 100µmol/L HgCl2. The peanut

shoots showed a very rapid response to mercury; leaves of

non-transgenic plants began to wilt two days after culture ini-

tiation 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 vigo-

rously and showed no difference in number or length com-

pared 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 contain-

ing 25 µmol/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 mer-

cury evolution rates in mercury-resistant plants were higher

than in non-transgenic control plants (Fig. 4). The mercury-

resistant lines X73-4-1 and X73-5-1 had a release rate 4 to 6

times higher than Georgia Runner and were significantly dif-

ferent from the control (P <0.01), while the line X73-3-3 that

failed the challenge of 100 µmol/L HgCl2 during rooting

7/31/2019 10310069_Yang



Figure 3. A) Test for resistance to mercury during rooting of somatic embryo-derived shoots. Left: Transgenic; right: Non-transgenic. B–C) Re-

generation 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: T1–11.

Figure 4. Elemental mercury [Hg(0)] release assays in transgenic peanut plants. In vitro -grown leaflets of independent transgenic lines were in-

cubated in liquid medium containing 25 µmol/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 differ-

ent 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, sepa-

rated 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.

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 re-

sistant 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.

Western blot analysis showed a correlation between mer-

cury resistance and the expression of mercuric ion reductase

in the leaf tissues. The line X73-3-3 that did not grow on mer-

cury-containing medium showed no MerA protein expression,

while the lines X73-4-1 and X73-5-1 that survived the chal-

lenge of 100µmol/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 pre-

sumed 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 pro-

cessing is occurring in the peanut leaves.

Test of merA as a potential selectable marker gene fortransformation in an organogenic system

The results of Northern blot, Western blot, and mercury vapor

assays clearly showed that the merA gene was expressed at

7/31/2019 10310069_Yang



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 de-scribed 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 ex-

plants containing the transgene remained green and pro-

duced new shoots while those that did not contain the trans-

gene turned brown and died (Fig.3B–C).

Discussion

In an effort to explore the development of an alternative se-

lectable marker for peanut transformation, we have studied

the expression of a codon-modified bacterial merA gene un-der the control of a plant regulatory DNA sequence, the Ara-

bidopsis 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, diver-

gent 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 ex-

pression 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 inflores-

cence, 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 couldbe 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 Arabidop-

sis gene that is strongly induced by auxin treatment (Kanda-

samy 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 ex-

pressed 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 ele-

ment for marker genes in a transformation system where se-

lection takes place post-embryonically or during organogene-

sis. Such Agrobacterium -mediated transformation systemsthat utilize organogenesis have been reported for peanut, al-

though 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 re-

sponse 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 Pea-

nut Foundation. We thank Evelyn Perry and Anne Bell for technical

assistance and Rich Meagher for providing promoters and experti-

se/facilities for mercury vapor analysis.

References

An YQ, McDowell JM, Huang S, McKinney EC, Chambliss S, Meagher

RB (1996) Strong, constitutive expression of the Arabidopsis ACT2/

ACT8 actin subclass in vegetative tissues. Plant J 10: 107–121

Cheng M, Jarret RL, Li Z, Xing A, Demski JW (1996) Production of fer-tile transgenic peanut (Arachis hypogaea L.) plants using Agro-

bacterium tumefaciens . Plant Cell Rep 15: 653–657

Chomczynski P (1992) One-hour downward alkaline capillary transfer

for blotting of DNA and RNA. Anal Biochem 201: 134–139

Church GM, Gilbert W (1984) Genomic sequencing. Proc Natl Acad

Sci USA 81: 1991–1995

Eapen S, George L (1994) Agrobacterium tumefaciens mediated

gene transfer in peanut (Arachis hypogaea L.) Plant Cell Rep 13:

582–586

Ebinuma H, Sugita K, Matsunaga E, Yamakado M (1997) Selection of

marker free transgenic plants using the isopentenyl transferase

gene. Proc Natl Acad Sci USA 94: 2117–2121

Endo S, Kasahara T, Sugita K, Matsunaga E, Ebinuma H (2001) The

isopentenyl transferase gene is effective as a selectable marker

gene for plant transformation in tobacco (Nicotiana tabacum cv.

Petite Havana SRI). Plant Cell Rep 7: 60–66

Fox B, Walsh CT (1982) Mercuric reductase. Purification and charac-

terization of a transposon-encoded flavoprotein containing an oxi-

dation-reduction-active disulfide. J Biol Chem 257: 2498–2503

Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of sus-

pension cultures of soybean root cells. Exp Cell Res 50: 150–158

Garbarino JE, Belknap WR (1994) Isolation of a ubiquitin-ribosomal

protein gene (ubi3 ) from potato and expression of its promoter in

transgenic plants. Plant Mol Biol 24: 119–127

7/31/2019 10310069_Yang



Garbarino JE, Rockhold DR, Belknap WR (1992) Expression of stress-

responsive ubiqutin genes in potato tubers. Plant Mol Biol 20:

235–244

Gough KC, Hawes WS, Kilpatrick J (2001) Cyanobacterial GR6 gluta-

mate-1-semialdehyde aminotransferase: a novel enzyme-based

selectable marker for plant transformation. Plant Cell Rep 20: 296–

300

Haldrup A, Peterson SG, Okkels FT (1998) Positive selection: a plantselection principle based on xylose isomerase, an enzyme used in

the food industry. Plant Cell Rep 18: 76–81

Huang S, An YQ, McDowell JM, McKinney EC, Meagher RB (1997)

The Arabidopsis ACT11 actin gene is strongly expressed in tissues

of the emerging inflorescence, pollen, and developing ovules.

Plant Mol Biol 33: 125–139

Joersbo M, Donaldson I, Kreiberg J, Petersen SG, Brunstedt J, Okkels

FT (1998) Analysis of mannose selection used for transformation of

sugar beet. Mol Breed 4: 111–117

Kandasamy MK, Gilliland LU, McKinney EC, Meagher RB (2001) One

plant actin isovariant, ACT7, is induced by auxin and required for

normal callus formation. Plant Cell 13: 1541–1554

Knapp JE, Chandle JM (1996) RNA/DNA miniprep from a single sam-

ple of orchid tissue. Biotechniques 20: 54–56Lee BT, Matheson NK (1984) Phosphomannoseisomerase and phos-

phoglucoisomerase in seeds of Cassia coluteoides and some

other legumes that synthesize galactomannan. Phytochemistry 23:

983–987

Li Z, Jarret RL, Pittman RN, Demski JW (1994) Shoot organogenesis

from cultured seed explants of peanut (Arachis hypogaea L.) using

thidiazuron. In Vitro Cell Dev Biol Plant 30: 187–191

Livingstone DM, Birch RG (1999) Efficient transformation and regen-

eration of diverse cultivars of peanut (Arachis hypogaea L.) by par-

ticle bombardment into embryogenic callus produced from mature

seeds. Mol Breed 5: 43–51

Lucca P, Ye X, Potrykus I (2001) Effective selection and regeneration

of transgenic rice plants with mannose as selective agent. Mol

Breed 7: 43– 49

Magbanua ZV, Wilde HD, Roberts JK, Chowdhury K, Abad J, Moyer

JW, Wetzstein HY, Parrott WA (2000) Field resistance to tomato

spotted wilt virus in transgenic peanut (Arachis hypogaea L.) ex-

pressing an antisense nucleocapsid gene sequence. Mol Breed 6:

227–236

McElroy D, Rothenberg M, Reece KS, Wu R (1990) Characterization of

the rice (Oryza sativa ) actin gene family. Plant Mol Biol 15: 257–268

McKently AH (1991) Direct somatic embryogenesis from axes of ma-

ture peanut embryos. In Vitro Cell Dev Biol Plant 27: 197–200

Mindlin SZ, Bass IA, Bogdanova ES, Gorlenko ZM, Kalyaeva ES, Pet-

rova MA, Nikiforov VG (2002) Horizontal transfer of mercury resist-

ance genes in environmental bacterial populations. Mol Biol 36:

160–170

Murashige T, Skoog F (1962) A revised medium for rapid growth and

bioassay with tobacco tissue culture. Physiol Plant 15: 473–497

Murray HG, Thompson WF (1980) Rapid isolation of high molecular

weight plant DNA. Nucl Acids Res 8: 4321–4325

Ozias-Akins P (1989) Plant regeneration from immature embryos of

peanut. Plant Cell Rep 8: 217– 218

Ozias-Akins P, Gill R (2001) Progress in the development of tissue cul-

ture and transformation methods applicable to the production of

transgenic peanut. Peanut Sci 28: 123–131

Ozias-Akins P, Schnall JA, Anderson WF, Singit C, Clemente TE,

Adang MJ, Weissinger AK (1993) Regeneration of transgenic pea-

nut plants from stably transformed embryogenic callus. Plant Sci

93: 185–194

Pike R, Lucas V, Stapleton P, Gilthorpe MS, Roberts G, Rowbury R,

Richards H, Mullany P, Wilson M (2002) Prevalence and antibiotic

resistance profile of mercury-resistant oral bacteria from children

with and without mercury amalgam fillings. J Antimicrobial Chemo-

therapy 49: 777–783

Reed J, Privalle L, Powell ML, Meghji M, Dawson J, Dunder E, Suttie J,

Wenck A, Launis K, Kramer C, Chang Y-F, Hansen G, Wright M

(2001) Phosphomannose isomerase: An efficient selectable marker

for plant transformation. In Vitro Cell Dev Biol Plant 37: 127–132

Rugh CL, Wilde HD, Stack NM, Thompson DM, Summers AO, Meag-

her RB (1996) Mercuric ion reduction and resistance in transgenic

Arabidopsis thaliana plants expressing a modified bacterial merA

gene. Proc Natl Acad Sci USA 93: 3182–3187

Rugh CL, Senecoff JF, Meagher RB, Merkle SA (1998) Development of

transgenic yellow poplar for mercury phytoremediation. Nature Bio-

tech 16: 925– 928

SAS Institute Inc (2000) SAS/C OnlineDocTM, Release 7.00, Cary, NC

Schowalter DB, Sommer SS (1989) The generation of radiolabeled

DNA and RNA probes with polymerase chain reaction. Anal Bio-chem 177: 90–94

Sharma KK, Anjaiah V (2000) An efficient method for the production of

transgenic plants of peanut (Arachis hypogaea L.) through Agro-

bacterium tumefaciens -mediated genetic transformation. Plant Sci

159: 7–19

Singsit C, Adang MC, Lynch RE, Anderson WA, Wang A, Cardineau

G, Ozias-Akins P (1997) Expression of a Bacillus thuringiensis

cryIAc gene in transgenic peanut plants and its efficacy against

lesser cornstalk borer. Transgen Res 6: 169–176

Sugita K, Matsunaga E, Ebinuma H (1999) Effective selection system

for generating marker-free transgenic plants independent of sexual

crossing. Plant Cell Rep 18: 941–947

Summers AO (1986) Organization, expression, and evolution of genes

for mercury resistance. Annu Rev Microbiol 40: 607–634

Thangavelu M, Belostotsky D, Bevan MW, Flavell RB, Rogers HJ,

Lonsdale DM (1993) Partial characterization of the Nicotiana taba-

cum actin gene family: evidence for pollen specific expression of

one of the gene family members. Mol Gen Genet 240: 290–295

Thomson JA (2001) Horizontal transfer of DNA from GM crops to bac-

teria and to mammalian cells. J Food Sci 66: 188–193

Wang AM, Fan H, Singsit C, Ozias-Akins P (1998) Transformation of

peanut with a soybean vspB promoter-uidA chimeric gene. I. Opti-

mization of a transformation system and analysis of GUS ex-

pression in primary transgenic tissue and plants. Physiol Plant 102:

38–48

Wright M, Dawson J, Dunder E, Suttie J, Reed J, Kramer C, Chang Y,

Novitzky R, Wang H, Moore LA (2001) Efficient biolistic transforma-tion of maize (Zea mays L.) and wheat (Triticum aestivum L.) using

the phosphomannose isomerase gene, pmi, as the selectable

marker. Plant Cell Rep 20: 429–436

Yang HY, Singsit C, Wang A, Gonsalves D, Ozias-Akins P (1998)

Transgenic peanut plants containing a nucleocapsid protein gene

of tomato spotted wilt virus show divergent levels of gene ex-

pression. Plant Cell Rep 17: 693–699

Zhang P, Potrykus I, Puonti-Kaerlas J (2000) Sufficient production of

transgenic cassava using negative and positive selection. Trans-

genic Res 9: 405–415

Documents

10310069_Yang